Compositions and Methods for Enhancing Bioenergetic Status in Female Germ Cells

ABSTRACT

Compositions and methods comprising bioenergetic agents for restoring the quality of aged oocytes, enhancing oogonial stem cells or improving derivatives thereof (e.g., cytoplasm or isolated mitochondria) for use in fertility-enhancing procedures, are described.

This application contains subject matter that may be related to U.S.provisional patent application Ser. Nos. 61/475,561, filed Apr. 14,2011, 61/600,505, filed Feb. 17, 2012, and 61/502,588, filed Jun. 29,2011, and PCT Application No. PCT/US2012/033643, filed Apr. 13, 2012,the entire disclosures of each of which are incorporated herein byreference. All patents and publications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent patent and publication was specifically and individuallyindicated to be incorporated by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by National Institutes of Health GrantNo. NIH R37-AG012279. The government has certain rights to theinvention.

CROSS-REFERENCE TO RELATED SUBJECT MATTER

This application is a division of U.S. patent application Ser. No.13/447,083, filed Apr. 13, 2012, Allowed, which claims benefit under 35U.S.C. §119(e) of U.S. provisional application Ser. No. 61/502,840,filed Jun. 29, 2011, and U.S. provisional application Ser. No.61/600,529, filed Feb. 17, 2012. The entire disclosures of theaforementioned patent applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Since the early 1950s, clinical management of problems associated withovarian insufficiency and failure, including infertility due to aging orinsults, has been restricted by the belief that the pool of oocytes setforth at birth is not amenable to replacement or renewal (Zuckerman,Recent Prog Horm Res 1951 6: 63-108). In other words, any therapeuticintervention had to conform to manipulation of the existing stockpile ofoocyte-containing follicles to produce a desired clinical outcome. In2004, however, studies with mice challenged the idea of a fixed ovarianreserve of oocytes being endowed at birth (Johnson et al., Nature 2004428: 145-150). Based on results from several experimental approaches, itwas concluded that ovaries of adult female mammals retain rare germlineor oogonial stem cells (OSCs) that routinely produce new oocytes in amanner analogous to germline stem cell support of sperm production inthe adult testis (Spradling, Nature 2004 428: 133-134). Several yearslater, OSCs were successfully isolated from neonatal and adult mouseovaries (Zou et al., Nat Cell Biol 2009 11: 631-636; Pachiarotti et al.,Differentiation 2010 79: 159-170). Collectively, these investigations,along with several other reports from studies of mice (Johnson et al.,Cell 2005 122: 303-315; Wang et al., Cell Cycle 2010 9: 339-349; Niikuraet al., Aging 2010 2: 999-1003) have conceptually validated the use ofOSCs as agents for transplantation and as targets for new therapies tomodulate ovarian function and female fertility (Tilly et al., BiolReprod 2009 80: 2-12; Tilly et al., Mol Hum Reprod 2009; 15: 393-398).In addition, the identification of dormant OSCs in atrophic ovaries ofaged mice, which spontaneously resume oocyte formation when exposed to ayoung adult ovarian environment, indicates that ovarian aging may bereversible (Niikura et al., Aging 2009 1: 971-978; Massasa et al., Aging2010; 2:1-2). The clinical utility of OSCs is now further confirmed byevidence shown herein that a comparable population of oocyte-producingstem cells exists in, and can be purified from, ovaries of healthyreproductive-age women.

Although these new studies indicate that oocyte numbers in adult ovariesare amenable to therapeutic expansion through OSC-based technology,ovarian aging and failure is determined by both a decline in oocytenumber as well as a decline in the quality of the oocytes present in theovaries. Hence, it is imperative to identify methods for improvingoocyte quality, especially in women of advancing maternal age. Duringthe past few decades, because of cultural and social changes, women inthe developed world have significantly delayed childbirth. For example,first birth rates for women 35-44 years of age in the United States haveincreased by more than 8-fold over the past 40 years (Ventura VitalHealth Stat 2009 47: 1-27; Matthews, NCHS Data Brief2009 21: 1-8). It iswell known that pregnancy rates in women at 35 or more years of age aresignificantly lower, both naturally and with assisted reproduction. Thedecline in live birth rate reflects a decline in response to ovarianstimulation by gonadotropin hormones (follicle-stimulating hormone orFSH, and luteinizing hormone or LH), reduced oocyte and embryo qualityand pregnancy rates, and an increased incidence of miscarriages andfetal aneuploidy. In fact, aging-associated chromosomal and meioticspindle abnormalities in eggs are considered the major factorsresponsible for the increased incidence of infertility, fetal loss(miscarriage) and conceptions resulting in birth defects—most notablytrisomy 21 or Down syndrome—in women at advanced reproductive ages(Henderson et al., Nature 1968 218: 22-28; Hassold et al., Hum Genet1985 70: 11-17; Battaglia et al., Hum Reprod 1996 11: 2217-2222; Hunt etal., Trends Genet 2008 24: 86-93). Although the occurrence andconsequences of aging-related aneuploidy in oocytes of humans and animalmodels have been extensively studied (Tarin et al., Biol Reprod 2001 65:141-150; Pan et al., Dev Biol 2008 316: 397-407; Duncan et al., BiolReprod 2009 81: 768-776), approaches to maintain fidelity of chromosomesegregation during meiotic cell division with age have remained elusive.At present there is no known intervention to improve the pregnancyoutcome of older female patients. In animal studies, chronicadministration of pharmacologic doses of anti-oxidants during thejuvenile period and throughout adult reproductive life has been reportedto improve oocyte quality in aging female mice (Tarin et al., Mol ReprodDev 2002 61: 385-397). However, this approach has significant long-termnegative effects on ovarian and uterine function, leading to higherfetal death and resorptions as well as decreased litter frequency andsize in treated animals (Tarin et al., Theriogenology 2002 57:1539-1550). Thus, clinical translation of chronic anti-oxidant therapythroughout reproductive life for maintaining or improving oocyte qualityin aging females is impractical.

Mitochondrial dysfunction has a major role in reproductive senescenceand, therefore, reproductive function in older women might be improvedby the use of mitochondrial nutrients (Bentov et al., Fertil Steril 201093: 272-275). Aging and age-related pathologies are frequentlyassociated with loss of mitochondrial function, due to decreasedmitochondrial numbers (biogenesis and mitophagy), increased aggregationof mitochondria, diminished mitochondrial activity (production of ATP,which is the main source of energy for cells) and mitochondrial membranepotential and/or accumulation of mitochondrial DNA (mtDNA) mutations anddeletions. As oocytes age and oocyte mitochondrial energy productiondecreases, many of the critical processes of oocyte maturation requiredto produce a competent egg, especially nuclear spindle activity andchromosomal segregation, become impaired (Bartmann et al., J AssistReprod Genet 2004 21: 79-83; Wilding et al., Zygote 2005 13: 317-23).Nicotinamide adenine dinucleotide (NAD⁺) is a small molecule regulatorof many other processes including signaling pathways, cell-cellcommunication, and epigenetic changes. Once thought to be very stable,levels of NAD⁺ rise in response to dieting and exercise. Increased NAD+levels are also associated with the diet known as caloric restriction(CR), which is known to delay numerous aspects of aging and diseases,including infertility (Sinclair Mech Ageing Dev 2005 26: 987; Selesniemiet al. Aging Cell 7: 622-629, 2008).

NAD⁺ levels are important for the proper function of mitochondria andthe cells that contain them. Cells with low mitochondrial NAD⁺ are proneto cell dysfunction and death (Yang et al., Cell 2008). Obesity andaging both reduce mitochondrial NAD⁺ levels, resulting in decreasedmitochondrial function, increased cell death, and an acceleration ofage-related diseases (Hafner et al. Aging 2010 2: 1-10). As oocytes ageand oocyte mitochondrial energy production decreases, many of theprocesses of oocyte maturation, especially meiotic spindle activity andchromosomal segregation, become impaired (Bartmann et al., J AssistReprod Genet 2004 21: 79-83; Wilding et al., Zygote 2005 13: 317-23).Raising NAD⁺ levels is a viable option for increasing the bioenergeticsand viability of cells, organs, tissues, and embryonic development.Downstream mediators include the sirtuin deaceylases (SIRT1-7) and thepoly-ADP ribose polymerases (PARPs). It is known to those skilled in theart that increasing NAD⁺ levels and boosting mitochondrial function canmimic the health benefits of caloric restriction (Yang et al., ExpGerontol 2006 41: 718-726).

The link between chronic anti-oxidant therapy for maintaining oocytequality in females of advanced reproductive age is established (Tarin etal., Hum Reprod 1995 10: 1563-1565) and data supporting a key role formitochondrial dysfunction in eggs as a driving force behind age-relatedfertility problems are available. For example, experimentally-inducedoxidative stress in isolated mouse oocytes reduces ATP levels, whichincreases meiotic spindle abnormalities leading to chromosomalmisalignment (Zhang et al., Cell Res 2006 16: 841-850). Additionally,while meiotic maturation of human oocytes can proceed over a range ofATP concentrations, oocytes with a higher ATP content show a muchgreater potential for successful embryogenesis, implantation anddevelopment (Van Blerkom et al., Hum Reprod 1995 10: 415-424).

Along these same lines, heterologous transfer of cytoplasmic extractsfrom young donor oocytes (viz. obtained from different women) into theoocytes of older women with a history of reproductive failure, aprocedure known as ooplasmic transplantation or ooplasmic transfer,demonstrated improved embryo development and delivery of live offspring.Unfortunately, however, the children born following this procedureexhibit mitochondrial heteroplasmy or the presence of mitochondria fromtwo different sources (Cohen et al., Mol Hum Reprod 1998 4: 269-280;Barritt et al., Hum Reprod 2001 16: 513-516; Muggleton-Harris et al.,Nature 1982 299: 460-462; Harvey et al., Curr Top Dev Biol 2007 77:229-249). This is consistent with the fact that maternally-derivedmitochondria present in the egg are used to “seed” the embryo withmitochondria, as paternally-derived mitochondria from the sperm aredestroyed shortly after fertilization (Sutovsky et al., Biol Reprod 200063: 582-590). Although the procedure involves transfer of cytoplasm andnot purified mitochondria from the donor eggs, the presence of donormitochondria in the transferred cytoplasm, confirmed by the passage of“foreign” mitochondria into the offspring, is widely believed to be thereason why heterologous ooplasmic transfer provides a fertility benefit(Harvey et al. Curr Top Dev Biol 2007 77: 229-249). Irrespective, thehealth impact of induced mitochondrial heteroplasmy in these children isas yet unknown; however, it has been demonstrated that a mouse model ofmitochondrial heteroplasmy produces a phenotype consistent withmetabolic syndrome (Acton et al., Biol Reprod 2007 77: 569-576).Arguably, the most significant issue with heterologous ooplasmictransfer is tied to the fact that mitochondria also contain geneticmaterial that is distinct from nuclear genes contributed by thebiological mother and biological father. Accordingly, the childrenconceived following this procedure have three genetic parents(biological mother, biological father, egg donor), and thus represent anexample of genetic manipulation of the human germline for the generationof embryos. Ooplasmic transplantation procedures that result inmitochondrial heteroplasmy are therefore now regulated and largelyprohibited by the FDA. For details, see CBER 2002 Meeting Documents,Biological Response Modifiers Advisory Committee minutes from May 9,2002, which are publically available from the FDA and “Letter toSponsors/Researchers—Human Cells Used in Therapy Involving the Transferof Genetic Material By Means Other Than the Union of Gamete Nuclei”,which is also publically available from the FDA on the worldwide web.While use of autologous mitochondria from somatic cells would avoidmitochondrial heteroplasmy, the somatic mitochondria are nonethelessinadequate, as they are prone to mitochondrial DNA damage and deletionsresulting in heritable mutations. Autologous sources of female germcells, namely OSCs and compositions obtained thereof (e.g., OSCcytoplasm or isolated mitochondria), in ooplasmic transplantationprocedures would prevent mitochondrial heteroplasmy, and alleviateethical and safety concerns currently associated with the procedure.Importantly, oocytes, which are prone to aging-associated defects, arenot of high enough quantity or quality to be reliably used in suchprocedures.

Accordingly, it is desirable to restore the quality of aged oocytes, aswell as to further enhance OSCs or improve derivatives thereof (e.g.,cytoplasm or isolated mitochondria) for use in conducting a range ofassisted reproductive technologies.

SUMMARY OF THE INVENTION

The present invention provides for the use of agents to enhancemitochondrial numbers, mitochondrial activity, cellular energy levels orcellular energy-producing potential (collectively referred to as“bioenergetic status”) in oocytes, postnatal female germline stem cells(also referred to herein as OSCs) and/or preimplantation embryos priorto conducting and/or following methods of in vitro fertilization, orfollowing exposure of ovaries, oocytes, OSCs and/or preimplantationembryos in vivo. In certain embodiments, agents for such uses includesoluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a Sirt1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof. These agents will becollectively referred to herein as “bioenergetic agents.”

In one aspect, the invention provides a composition containing one ormore of an oocyte, an oogonial stem cell (OSC) or the progeny of an OSC,and a bioenergetic agent (e.g., one or more of soluble precursors toNAD⁺ (e.g., tryptophan, quinolinic acid, nicotinamide mononucleotide,nicotinamide riboside, and nicotinic acid), fisetin, quercetin,resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone, metformin,apigenin, luteolin, tyrphostin-8, berberine, a CD38 inhibitor, SRT-1720,SIRT1 activator, a compound of any one of formulas I-XV, or functionalderivatives thereof).

In another aspect, the invention provides an isolated cell havingenhanced mitochondrial function, where the cell is one or more of anoocyte, an oogonial stem cell (OSC) or the progeny of an OSC, where thecell has been contacted with a bioenergetic agent (one or more ofsoluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, SIRT1 activator, a compound of any one of formulasI-XV, or functional derivatives thereof).

In yet another aspect, the invention provides a composition containingOSC mitochondria or oocyte mitochondria and a bioenergetic agent that isone or more of soluble precursors to NAD⁺ (e.g., tryptophan, quinolinicacid, nicotinamide mononucleotide, nicotinamide riboside, and nicotinicacid), fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator, a compound ofany one of formulas I-XV, or functional derivatives thereof.

In still another aspect, the invention provides an isolatedmitochondria, where the mitochondria has been contacted with abioenergetic agent that is any one or more of one or more of solubleprecursors to NAD⁺ (e.g., tryptophan, quinolinic acid, nicotinamidemononucleotide, nicotinamide riboside, and nicotinic acid), fisetin,quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone,metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof.

In another aspect, the invention provides an isolated cell-freecomposition containing OSC mitochondria or oocyte mitochondria and abioenergetic agent that any one or more of one or more of solubleprecursors to NAD⁺ (e.g., tryptophan, quinolinic acid, nicotinamidemononucleotide, nicotinamide riboside, and nicotinic acid), fisetin,quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone,metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof.

In another aspect, the invention provides a method of preparing anoocyte for in vitro fertilization (IVF), the method involvingtransferring a composition containing OSC mitochondria and abioenergetic agent that is one or more of any one or more of one or moreof soluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof, into an autologousoocyte, thereby preparing the oocyte for in vitro fertilization. In oneembodiment, the composition containing OSC mitochondria is a purifiedpreparation of mitochondria obtained from the OSC.

In yet another aspect, the invention provides an oocyte preparedaccording to the method of the previous aspect or any other aspect ofthe invention delineated herein. In yet another aspect, the inventionprovides a method of in vitro fertilization, the method involving thesteps of (a) incubating an OSC from a female subject with a bioenergeticagent that is any one or more of one or more of soluble precursors toNAD⁺ (e.g., tryptophan, quinolinic acid, nicotinamide mononucleotide,nicotinamide riboside, and nicotinic acid), fisetin, quercetin,resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone, metformin,apigenin, luteolin, tyrphostin-8, berberine, a CD38 inhibitor, SRT-1720,SIRT1 activator, a compound of any one of formulas I-XV, or functionalderivatives thereof; (b) obtaining a composition containing OSCmitochondria from the OSC; (c) transferring the composition into anisolated, autologous oocyte; and (d) fertilizing the autologous oocytein vitro to form a zygote. In one embodiment, the method furtherinvolves transferring a preimplantation stage embryo derived from thezygote, into the uterus of a female subject. In one embodiment, step a)is optional and step b) further involves incubating the compositioncontaining OSC mitochondria with a bioenergetic agent that is any one ormore of one or more of soluble precursors to NAD⁻ (e.g., tryptophan,quinolinic acid, nicotinamide mononucleotide, nicotinamide riboside, andnicotinic acid), fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator, a compound ofany one of formulas I-XV, or functional derivatives thereof.

In still another aspect, the invention provides a method of preparing anoocyte for in vitro fertilization, the method involving transferring acomposition containing oocyte mitochondria and a bioenergetic agent thatis any one or more of one or more of soluble precursors to NAD⁺ (e.g.,tryptophan, quinolinic acid, nicotinamide mononucleotide, nicotinamideriboside, and nicotinic acid), fisetin, quercetin, resveratrol, DOI,hydroxytyrosol, pyrroloquinoline quinone, metformin, apigenin, luteolin,tyrphostin-8, berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator,a compound of any one of formulas I-XV, or functional derivativesthereof, into an autologous oocyte, thereby preparing the oocyte for invitro fertilization. In one embodiment, the composition containingoocyte mitochondria is oocyte cytoplasm without a nucleus. In anotherembodiment, the composition containing oocyte mitochondria is a purifiedpreparation of mitochondria obtained from the oocyte. In oen embodiment,the method further involves fertilizing the oocyte in vitro to form azygote and transferring a preimplantation stage embryo derived from saidzygote, into the uterus of the female subject. In another embodiment,the zygote and pre-implantation stage embryo is incubated with abioenergetic agent selected from the group consisting of tryptophan,quinolinic acid, nicotinamide mononucleotide, nicotinamide riboside,nicotinic acid, fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a SIRT1 activator, a CD38 inhibitor, a compound of any one offormulas I-XV, and functional derivatives thereof.

In still another aspect, the invention provides an oocyte preparedaccording to the method of the previous aspect or any other aspect ofthe invention delineated herein.

In yet another aspect, the invention provides a method of in vitrofertilization, the method involving the steps of (a) incubating anoocyte from a female subject with a bioenergetic agent that is any oneor more of one or more of soluble precursors to NAD⁺ (e.g., tryptophan,quinolinic acid, nicotinamide mononucleotide, nicotinamide riboside, andnicotinic acid), fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator, a compound ofany one of formulas I-XV, or functional derivatives thereof; (b)obtaining a composition containing oocyte mitochondria from the oocyte;(c) transferring the composition into an isolated, autologous oocyte;and (d) fertilizing the autologous oocyte in vitro to form a zygote. Inone embodiment, the method further involves transferring apreimplantation stage embryo derived from the zygote, into the uterus ofa female subject. In another embodiment, step a) is optional and step b)further involves incubating the composition containing oocytemitochondria with a bioenergetic agent that is any one or more of one ormore of soluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof.

In still another aspect, the invention provides a method of in vitrofertilization, the method involving the steps of: incubating an oocytefrom a female subject with a bioenergetic agent that is any one or moreof one or more of soluble precursors to NAD⁺ (e.g., tryptophan,quinolinic acid, nicotinamide mononucleotide, nicotinamide riboside, andnicotinic acid), fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator, a compound ofany one of formulas I-XV, or functional derivatives thereof; andfertilizing the oocyte in vitro to form a zygote.

In still another aspect, the invention provides a composition containinga solution selected from the group consisting of cell culture medium,oocyte retrieval solution, oocyte washing solution, oocyte in vitromaturation medium, ovarian follicle in vitro maturation medium, oocytein vitro fertilization medium, embryo culture medium, cleavage medium,vitrification solution, cryopreservation solution and embryo thawingmedium and a bioenergetic agent that is any one or more of one or moreof soluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof.

In still another aspect, the invention provides a method of improvingfertility in a female subject, the method containing administering tothe subject a bioenergetic agent that is any one or more of one or moreof soluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof, in an amount effectiveto improve oocyte and/or OSC quality, de novo production and/or ovulatedoocyte yield, thereby improving fertility in the female subject. In oneembodiment, the bioenergetic agent is systemically administered to thefemale subject. In another embodiment, the bioenergetic agent is locallyadministered to an ovary of the female subject. In yet anotherembodiment, the pregnancy outcomes of the female subject are improvedcompared to a reference standard.

In still another aspect, the invention provides a method of in vitrofertilization, the method containing the steps of:

(a) administering to a female subject a bioenergetic agent that is anyone or more of one or more of soluble precursors to NAD⁺ (e.g.,tryptophan, quinolinic acid, nicotinamide mononucleotide, nicotinamideriboside, and nicotinic acid), fisetin, quercetin, resveratrol, DOI,hydroxytyrosol, pyrroloquinoline quinone, metformin, apigenin, luteolin,tyrphostin-8, berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator,a compound of any one of formulas I-XV, or functional derivativesthereofin an amount effective to improve oocyte and/or OSC de novoproduction, quality and/or ovulated oocyte yield;

(b) obtaining an oocyte from the female subject; and

(c) fertilizing the oocyte in vitro to form a zygote. In one embodiment,the bioenergetic agent is systemically administered to the femalesubject. In another embodiment, the bioenergetic agent is locallyadministered to an ovary of the female subject. In another embodiment,step a) is conducted prior to steps b) and c) and/or after steps b) andc). In another embodiment, the method further involving step d)transferring a preimplantation stage embryo derived from the zygote,into the uterus of the female subject and continuing to administer tothe female subject the bioenergetic agent. In another embodiment, stepb) and/or step c) further contains incubating the oocyte with abioenergetic agent that is any one or more of one or more of solubleprecursors to NAD⁺ (e.g., tryptophan, quinolinic acid, nicotinamidemononucleotide, nicotinamide riboside, and nicotinic acid), fisetin,quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone,metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof.

In another embodiment, the pregnancy outcomes of the female subject areimproved compared to a reference standard.

In still another aspect, the invention provides a method of sustainingembryonic development in a pregnant female subject in need thereof, themethod containing administering to the subject a therapeuticallyeffective amount of a bioenergetic agent that is any one or more of oneor more of soluble precursors to NAD⁺ (e.g., tryptophan, quinolinicacid, nicotinamide mononucleotide, nicotinamide riboside, and nicotinicacid), fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator, a compound ofany one of formulas I-XV, or functional derivatives thereof, therebysustaining embryonic development in the pregnant female subject. In oneembodiment, the bioenergetic agent is systemically administered to thefemale subject. In another embodiment, the bioenergetic agent is locallyadministered to an ovary of the female subject.

In still another aspect, the invention provides a method of restoringovarian function in a female subject in need thereof, containingadministering a therapeutically effective amount of a bioenergetic agentthat is any one or more of one or more of soluble precursors to NAD⁺(e.g., tryptophan, quinolinic acid, nicotinamide mononucleotide,nicotinamide riboside, and nicotinic acid), fisetin, quercetin,resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone, metformin,apigenin, luteolin, tyrphostin-8, berberine, a CD38 inhibitor, SRT-1720,a SIRT1 activator, a compound of any one of formulas I-XV, or functionalderivatives thereof, thereby restoring ovarian function in the femalesubject.

In one embodiment, the bioenergetic agent is systemically administeredto the female subject. In another embodiment, the bioenergetic agent islocally administered to an ovary of the female subject. In anotherembodiment, where the female subject has premature ovarian failure.

In still another aspect, the invention provides a method of preparing atissue or cell thereof from a female subject for harvest, the methodcontaining administering an effective amount of a bioenergetic agentthat is any one or more of one or more of soluble precursors to NAD⁺(e.g., tryptophan, quinolinic acid, nicotinamide mononucleotide,nicotinamide riboside, and nicotinic acid), fisetin, quercetin,resveratrol, DOI, hydroxytyrosol, pyrroloquinoline quinone, metformin,apigenin, luteolin, tyrphostin-8, berberine, a CD38 inhibitor, SRT-1720,a SIRT1 activator, a compound of any one of formulas I-XV, or functionalderivatives thereof to the female subject, thereby preparing the tissueor cell thereof from the female subject for harvest. In one embodiment,the tissue is ovary, ovarian follicle, bone marrow or peripheral blood.

In still another aspect, the invention provides a method of producing anoocyte, containing culturing a stem cell that is an OSC, embryonic stemcell, pancreatic stem cell, skin stem cell or induced pluripotent stemcell (iPS cell) in the presence of a bioenergetic agent that is any oneor more of one or more of soluble precursors to NAD⁺ (e.g., tryptophan,quinolinic acid, nicotinamide mononucleotide, nicotinamide riboside, andnicotinic acid), fisetin, quercetin, resveratrol, DOI, hydroxytyrosol,pyrroloquinoline quinone, metformin, apigenin, luteolin, tyrphostin-8,berberine, a CD38 inhibitor, SRT-1720, a SIRT1 activator, a compound ofany one of formulas I-XV, or functional derivatives thereof, underconditions sufficient to differentiate the stem cell into an oocyte.

In another aspect, the invention features a composition containing anisolated cell that is an oocyte, an oogonial stem cell (OSC) or theprogeny of an OSC and a bioenergetic agent for use in in vitrofertilization.

In another aspect, the invention features an isolated cell havingenhanced mitochondrial function relative to a reference, where the cellis an oocyte, an oogonial stem cell (OSC) or the progeny of an OSC, andwhere the cell has been contacted with a bioenergetic agent for use inin vitro fertilization.

In another aspect, the invention features a bioenergetic agent that istryptophan, quinolinic acid, nicotinamide mononucleotide, nicotinamideriboside, nicotinic acid, fisetin, quercetin, resveratrol, DOI,hydroxytyrosol, pyrroloquinoline quinone, metformin, apigenin, luteolin,tyrphostin-8, berberine, CD38 inhibitor, a compound of any one offormulas I-XV, or functional derivatives thereof for use in one or moreof improving the fertility of a female, sustaining embryonic developmentin a pregnant female, restoring or increasing ovarian function in afemale, preparing a tissue or cell thereof from a female for harvest orpreparing an oocyte.

In various embodiments of any of the above aspects or any aspect of theinvention delineated herein, the bioenergetic agent is one or more ofsoluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, resveratrol, DOI, hydroxytyrosol, pyrroloquinolinequinone, metformin, apigenin, luteolin, tyrphostin-8, berberine, a CD38inhibitor, SRT-1720, a SIRT1 activator, a compound of any one offormulas I-XV, or functional derivatives thereof. In various embodimentsof any of the above aspects or any aspect delineated herein, thebioenergetic agent is a compound shown in FIG. 29.

In various embodiments of any of the above aspects or any aspectdelineated herein, a composition of the invention further contains asolution selected from one or more of cell culture medium, oocyteretrieval solution, oocyte washing solution, oocyte in vitro maturationmedium, ovarian follicle in vitro maturation medium, oocyte in vitrofertilization medium, vitrification solution and cryopreservationsolution. In various embodiments, the composition contains ovariantissue, ovarian follicles, bone marrow, umbilical cord blood orperipheral blood. In other embodiments, the OSC is an isolatednon-embryonic stem cell that is mitotically competent and expresses oneor more of Vasa, Oct-4, Dazl, Stella and optionally a stage-specificembryonic antigen. In other embodiments, the OSC is obtained fromovarian tissue. In various embodiments, the OSC is obtained from anon-ovarian tissue. In particular embodiments, the non-ovarian tissue isblood or bone marrow. In various embodiments, the cell is an ovarianstem cell, where the cell has been contacted with a bioenergetic agent.In other embodiments, the contacted cell has increased mitochondrial DNAcopy number and/or increased ATP-generating capacity. In still otherembodiments, the number of mitochondria is increased by about 10%, 20%,30%, 40%, 50% or 60%. In various embodiments of any of the above aspectsor any aspect delineated herein, increased mitochondrial function isdetected by assaying mtDNA content, ATP, NAD+/NADH, mitochondrial mass,membrane potential, and gene expression of known mitochondrial massregulators and electron transport chain components.

In various embodiments of any of the above aspects or any aspectdelineated herein, the cell is in a solution that is any one or more ofcell culture medium, oocyte retrieval solution, oocyte washing solution,oocyte in vitro maturation medium, ovarian follicle in vitro maturationmedium, oocyte in vitro fertilization medium, vitrification solution andcryopreservation solution. In other embodiments, the mitochondria is ina cell that is one or more of an oocyte, an oogonial stem cell (OSC) orthe progeny of an OSC. In particular embodiments, cell is in a mixturewith ovarian tissue, ovarian follicles, bone marrow, umbilical cordblood or peripheral blood.

In various embodiments of any of the above aspects or any aspectdelineated herein, the composition containing OSC mitochondria is OSCcytoplasm without a nucleus. In various embodiments, the compositioncontaining oocyte mitochondria is oocyte cytoplasm without a nucleus. Invarious embodiments, the composition containing OSC mitochondria is apurified preparation of mitochondria obtained from the OSC. In otherembodiments, the composition containing an oocyte mitochondria is apurified preparation of mitochondria obtained from the oocyte.

In other embodiments of any of the above aspects or any aspectdelineated herein, the mitochondria is in a cell that is one or more ofan oocyte, an oogonial stem cell (OSC) or the progeny of an OSC, and abioenergetic agent. In various embodiments of any of the above aspectsor any aspect delineated herein, the cell is in a mixture with ovariantissue, ovarian follicles, bone marrow, umbilical cord blood orperipheral blood. In still other embodiments, the pregnancy outcomes ofthe female subject are improved compared to a reference standard. Instill other embodiments, the bioenergetic agent is systemicallyadministered to the female subject or is locally administered to anovary of the female subject.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims. Thus, other aspects of theinvention are described in the following disclosure and are within theambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, incorporatedherein by reference.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the U.S. Patent and TrademarkOffice upon request and payment of necessary fees.

FIG. 1 depicts validation of a fluorescence activated cell sorting(FACS)-based protocol for OSC isolation. In FIG. 1a , immunofluorescenceanalysis of VASA expression (green with blue DAPI counterstain) is shownin adult mouse ovaries using antibodies against the NH₂ or COOH terminusof VASA (scale bars, 50 μm). In FIG. 1b , immunomagnetic sorting ofdispersed mouse ovaries or isolated oocytes is shown using antibodiesagainst the NH₂ or COOH terminus of VASA. Fraction 1 contains cells plusbeads prior to separation, Fraction 2 is a wash or flow-through fraction(non-immunoreactive) and Fraction 3 is a bead fraction (VASA-positivecells, highlighted by white arrows). In FIG. 1c , FACS analysis of liveor permeabilized cells from dispersed mouse ovaries using antibodiesagainst the NH₂ or COOH terminus of VASA is shown. Viable VASA-positivecells are only detected with the COOH antibody (red dashed box) whereaspermeabilization enables isolation of VASA-positive cells using the NH₂antibody (blue dashed box). In FIG. 1d , permeabilization of viableVASA-positive cells (red dashed box) obtained with the COOH antibodyenables re-isolation of the same cells by FACS using the NH₂ antibody(blue dashed box). In FIG. 1e , a schematic representation of the FACSprotocols employed using the VASA-COOH antibody for isolation of viableOSCs is shown. FIG. 1f depicts gene expression analysis of germlinemarkers Blimp1 (also referred to as PR domain containing 1 with ZNFdomain or Prdm1), Stella, Fragilis (also referred to as interferoninduced transmembrane protein 3 or Ifitm3), Tert (telomerase reversetranscriptase), Vasa, Dazl (deleted in azoospermia like) and oocytemarkers Nobox (newborn ovary homeobox), Zp3 (zona pellucida glycoprotein3), Gdf9 (growth differentiation factor 9) in each cell fractionproduced during the ovarian dispersion process to obtain cells forFACS-based isolation of OSCs using the VASA-COOH antibody (+ve,VASA-positive viable cell fraction after FACS; −ve, VASA-negative viablecell fraction after FACS; No RT, PCR of RNA sample without reversetranscription; (3-actin, sample loading control).

FIG. 2 depicts OSC fractions isolated from adult mouse ovaries byimmunomagnetic bead sorting that contain contaminating oocytes. Geneexpression analysis of germline markers (Blimp 1, Stella, Fragilis,Tert, Vasa, Dazl) and oocyte-specific markers (Nobox, Zp3, Gdf9) isshown in young adult mouse ovaries (positive control) or the final cellfraction obtained following VASA-COOH antibody-based immunomagnetic beadsorting of dispersed young adult mouse ovaries (No RT, PCR of sortedcell RNA sample without reverse transcription; (β-actin, sample loadingcontrol).

FIG. 3 depicts isolation of VASA-positive cells from adult mouse andhuman ovaries using FACS. In FIG. 3a and b , the representativehistological appearance of adult ovarian tissue used for human (a) andmouse (b) OSC isolation is shown. Scale bars, 100 μm. In FIGS. 3c and d, the morphology of viable cells isolated by FACS based on cell-surfaceexpression of VASA is shown. Scale bars, 10 μm. FIG. 3e provides thegene expression profile of starting ovarian material andfreshly-isolated OSCs, showing assessment of 3 different patients asexamples for human tissue analysis (No RT: PCR of RNA sample withoutreverse transcription; β-actin, sample loading control). In FIG. 3fthrough FIG. 3k , a teratoma formation assay showing an absence oftumors in mice 24 weeks after receiving injections of mouse OSCs (3 f)compared with development of tumors in mice 3 weeks after injection ofmouse embryonic stem cells (ESCs)_is shown (FIG. 3g through FIG. 3j ;panels 3 h through 3 j show examples of cells from all three germlayers, with neural rosette highlighted in panel 3 h, inset), along witha summary of the experimental outcomes (3 k).

FIG. 4 depicts functional eggs obtained from mouse OSCs afterintraovarian transplantation. In FIGS. 4a and 4b , examples of growingfollicles containing GFP-negative and GFP-positive (brown against a bluehematoxylin counterstain) oocytes are shown in ovaries of wild-type miceinjected with GFP-expressing OSCs 5-6 months earlier. In FIG. 4c ,examples of ovulated GFP-negative eggs (in cumulus-oocyte complexes),and resultant embryos (2-cell, 4-cell, compact morula (CM) and earlyblastocyst (EB) stage embryos are shown as examples) generated by IVFare shown, following induced ovulation of wild-type female mice thatreceived intraovarian transplantation of GFP-expressing OSCs 5-6 monthsearlier. In FIGS. 4d and 4e , examples of GFP-positive eggs (incumulus-oocyte complexes) obtained from the oviducts are shown followinginduced ovulation of wild-type female mice that received intraovariantransplantation of GFP-expressing OSCs 5-6 months earlier. These eggswere in-vitro fertilized using wild-type sperm, resulting in 2-cellembryos that progressed through preimplantation development (examples ofGFP-positive embryos at the 2-cell, 4-cell, 8-cell, compacted morula(CM), expanded morula (EM), blastocyst (B) and hatching blastocyst (HB)stage are shown) to form hatching blastocysts 5-6 days afterfertilization.

FIG. 5 depicts germ cell colony formation by mouse and human OSCs invitro. Immunofluorescence-based analysis of VASA expression is shown inFIGS. 5b and 5d ; (green with blue DAPI counterstain) in typical germcell colonies formed by mouse (5 a, 5 b) and human (5 c, 5 d) OSCs afterestablishment on mouse embryonic fibroblasts (MEFs) in vitro (typicalcolonies are highlighted by white dashed lines).

FIG. 6 depicts evaluation of mouse and human ovary-derived VASA-positivecells in defined cultures. FIGS. 6a through 6d show assessment of OSCproliferation by dual detection of VASA expression (green) and BrdUincorporation (red) in mouse (6 a, 6 b) and human (6 c, 6 d) OSCsmaintained in MEF-free cultures. FIG. 6e shows the typical growth curvefor MEF-free cultures of mouse OSCs after passage and seeding 2.5×10⁴cells per well in 24-well culture plates. FIG. 6f shows FACS analysisusing the COOH antibody to detect cell-surface expression of VASA inmouse OSCs after months of propagation (example shown, passage 45). FIG.6g indicates the gene expression profile of starting ovarian materialand cultured mouse and human OSCs after 4 or more months of propagationin vitro (No RT, PCR of RNA sample without reverse transcription;β-actin, sample loading control). Two different human OSC lines (OSC1and OSC2) established from two different patients are shown as examples.FIGS. 6h and 6i show representative immunofluorescence analysis ofBLIMP1, STELLA and FRAGILIS expression (green) in mouse (h) and human(i) OSCs in MEF-free cultures. Cells were counterstained with DAPI(blue) and rhodamine-phalloidin (red) to visualize nuclear DNA andcytoplasmic F-actin, respectively.

FIG. 7 depicts spontaneous oogenesis from cultured mouse and human OSCs.FIGS. 7a through 7c provide examples of immature oocytes formed by mouseOSCs in culture, as assessed by morphology (7 a), expression of oocytemarker proteins VASA and KIT (7 b); note cytoplasmic localization ofVASA), and the presence of mRNAs encoding the oocyte marker genes Vasa,Kit, Msy2 (also referred to as Y box protein 2 or Ybx2), Nobox, Lhx8,Gdf9, Zp1, Zp2 and Zp3 (7 c); No RT: PCR of RNA sample without reversetranscription; β-actin, sample loading control). Scale bars, 25 μm. FIG.7d indicates the number of immature oocytes formed by mouse OSCs 24, 48and 72 hours after passage and seeding 2.5×10⁴ cells per well in 24-wellculture plates (culture supernatants were collected at each time pointfor determination, and thus the values represent numbers generated overeach 24 hour block, not cumulative numbers; mean±SEM, n=3 independentcultures). FIGS. 7e through 7g show in-vitro oogenesis from human OSCs,with examples of immature oocytes formed by human OSCs in culture (7 f,morphology; 7 g, expression of oocyte marker proteins VASA, KIT, MSY2and LHX8) and numbers formed following passage and seeding of 2.5×10⁴cells per well in 24-well culture plates (7 e; mean±SEM, n=3 independentcultures) shown. The presence of mRNAs encoding oocyte marker genes(Vasa, Kit, Msy2, Nobox, Lhx8, Gdf9, Zp1, Zp2, Zp3) in human OSC-derivedoocytes is shown in panel c along with results for mouse OSC-derivedoocytes. Scale bars, 25 μm. In FIG. 7h , immunofluorescence-baseddetection of the meiotic recombination markers, DMC1 (dosage suppressorof mckl homolog) and SYCP3 (synaptonemal complex protein 3) (red againstblue DAPI counterstain), is shown in nuclei of cultured human OSCs;human ovarian stromal cells served as a negative control. In FIG. 7i ,FACS-based ploidy analysis of cultured human OSCs is shown 72 hoursafter passage. Results from ploidy analysis of cultured humanfibroblasts (negative control) and cultured mouse OSCs are presented inFIG. 9.

FIG. 8 depicts the detection of oocyte-specific markers in adult humanovaries. Immunofluorescence analysis of VASA (8 a, red), KIT (8 b,green), MSY2 (8 c, red) and LHX8 (8 d, green) expression in oocytes inadult human ovarian cortical tissue is shown (see also FIG. 10h ).Sections were counterstained with DAPI (blue) for visualization ofnuclei. Scale bars, 25 μm.

FIG. 9 depicts ploidy analysis of human fibroblasts and mouse OSCs inculture. FIGS. 9a and 9b show representative FACS-based assessment ofploidy status in cultures of actively-dividing human fetal kidneyfibroblasts (9 a) and in mouse OSCs collected 48 hours after passage (9b). Haploid (1 n) cells were only detected in the germline cultures,consistent with results from analysis of human OSCs maintained in vitro(see FIG. 7i ), whereas all cultures contained diploid (2 n) andtetraploid (4 n) populations of cells.

FIG. 10 depicts generation of oocytes from human OSCs in human ovarytissue. Direct (live-cell) GFP fluorescence analysis of human ovariancortical tissue following dispersion, re-aggregation with GFP-hOSCs (10a) and in-vitro culture for 24-72 hours (10 b, 10 c) is shown. Note theformation of large single GFP-positive cells surrounded by smallerGFP-negative cells in compact structures resembling follicles (FIGS. 10band 10c ; scale bars, 50 μm). Examples of immature follicles containingGFP-positive oocytes (brown, highlighted by black arrowheads, against ablue hematoxylin counterstain) in adult human ovarian cortical tissueinjected with GFP-hOSCs and xenografted into NOD/SCID female mice areshown (FIG. 10d , 1 week post-transplant; FIG. 10f , 2 weekspost-transplant). Note comparable follicles with GFP-negative oocytes inthe same grafts. As negative controls, all immature follicles in humanovarian cortical tissue prior to GFP-hOSC injection and xenografting (10e) or that received vehicle injection (no GFP-hOSCs) prior toxenografting (10 g) contained GFP-negative oocytes after processing forGFP detection in parallel with the samples shown above. FIG. 10h showsdual immunofluorescence analysis of GFP expression (green) and eitherthe diplotene stage oocyte-specific marker MSY2 (red) or the oocytetranscription factor LHX8 (red) in xenografts receiving GFP-hOSCinjections. Note that GFP was not detected in grafts prior to GFP-hOSCinjection, whereas MSY2 and LHX8 were detected in all oocytes. Sectionswere counterstained with DAPI (blue) for visualization of nuclei. Scalebars, 25 μm.

FIG. 11 depicts morphometry-based assessment of oocyte formation inhuman ovarian xenografts following GFP-hOSC transplantation. The totalnumber of primordial and primary follicles in 3 randomly selected humanovarian cortical tissue samples (labeled 1, 2 and 3) are shown, 7 daysafter injecting GFP-hOSCs and xenografting into NOD/SCID mice, whichcontain GFP-negative (host-derived) or GFP-positive (OSC-derived)oocytes (see FIGS. 10d through 10g for examples).

FIG. 12 depicts cryopreservation and thawing of human ovarian corticaltissue and freshly-isolated human OSCs. FIGS. 12a and 12b show thehistological appearance of adult human ovarian cortical tissue beforeand after vitrification, highlighting the maintenance of tissueintegrity and the large numbers of oocytes (black arrowheads) thatsurvive the freeze-thaw procedure. In FIG. 12c , the percent cell lossfollowing freeze-thaw of freshly-isolated human OSCs is shown (resultsfrom two different patients).

FIG. 13 depicts an overview of an Autologous Germline MitochondrialEnergy Transfer procedure which is described in U.S. Patent ApplicationSer. No. 61/475,561, filed on Apr. 14, 2011, entitled “Compositions andMethods for Autologous Germline Mitochondrial Energy Transfer.” Notethat OSCs used as a source of mitochondria for the transfer, and the eggto be fertilized which will receive the mitochondria, are obtained fromthe same subject.

FIG. 14 depicts fluorescence activated cell sorting (FACS)-based germcell purification from bone marrow preparations of adult female miceduring estrus of the female reproductive cycle using cell surfaceexpression of Vasa to isolate the cells.

FIG. 15 depicts fluorescence activated cell sorting (FACS)-based germcell purification from peripheral blood preparations of adult femalemice during estrus of the female reproductive cycle using cell surfaceexpression of Vasa to isolate the cells.

FIG. 16 depicts prevention of the aging-related decline in ovulatedoocyte numbers as a result of restricted caloric intake (“CR”). (A)Yield and morphology of oocytes obtained after induced ovulation of3-mo-old (3M) ad-libitum (AL) diet (AL)-fed (n=6), 12-mo-old (12M)AL-fed (n=12), and 12M CR-AL-fed (n=6) mice (mean±SEM; *, P<0.05 vs. 3MAL-fed females). (B) Number of in-vitro fertilized metaphase stage II(MII) oocytes that developed to blastocysts per induced ovulation cycleper female (n=11-16 mice group; mean±SEM; *, P<0.05 vs. 3M AL-fedfemales). (C) Number of non-atretic immature follicles per ovary in 3MAL-fed, 12M AL-fed and 12M AL-CR-fed mice (mean±SEM, n=9-14 mice pergroup; *, P<0.05 vs. 3M AL-fed females; **, P<0.05).

FIG. 17 depicts the lack of effect of CR on preimplantation embryonicdevelopment following IVF. (A and B) Percent of cumulus cell-denuded MIIoocytes (A) or cumulus-enclosed oocytes (B) collected from 3M AL-fed,12M AL-fed and 12M CR-AL-fed female mice that developed to 2-cell stageembryos (2CE) following in-vitro fertilization, and the percent of 2CEor total inseminated oocytes (TIO) that developed to blastocyst stage(B) embryos [B(2CE) and B(TIO), respectively]. Data are the mean±SEM ofthe following: (A) n=55-140 denuded MII oocytes from 3 independentexperiments using a total of 6-9 mice per group; (B) n=38-144cumulus-oocyte complexes from 3 independent experiments using a total of5-7 mice per group.

FIG. 18 depicts the relationship between oocyte yield and body weight.(A and B) Assessment of body weight versus superovulated oocyte yield in3M AL-fed (A), 12M AL-fed (B) and 12M CR-AL-fed (C) females on amouse-by-mouse basis.

FIG. 19 depicts prevention of aging-associated aneuploidy in MII oocytesby CR. (A) Example of a hyperploid MII oocyte containing 21 chromosomes(DAPI staining of DNA shown in blue). (B) Incidence of hyperploidy,hypoploidy and premature sister chromatid separation (and totalchromosomal defects from all 3 endpoints combined) in MII oocytes of 3MAL-fed, 12M AL-fed and 12M CR-AL-fed females (mean ±SEM, n=18-23 matureoocytes analyzed per group in each experiment replicated 4 times using atotal of 20-34 mice per group; *, P<0.05 vs. 3M AL-fed females; nd, nonedetected).

FIG. 20 depicts prevention of spindle and chromosomal alignment defectsin oocytes of aged females by CR. (A and B) Incidence of spindleabnormalities (A) and chromosomal misalignment on the metaphase plate(B) in MII oocytes of 3M AL-fed, 12M AL-fed and 12M CR-AL-fed mice(mean±SEM, n=3-20 oocytes analyzed per group in each experimentreplicated 4-7 times using a total of 4-8 mice per group; *, P<0.05 vs.3M AL-fed females). (C) Representative examples of meiotic spindles inMII oocytes from the indicated mice (n=22-72 oocytes analyzed pergroup), after labeling with a-tubulin antibody (green) andcounterstaining of DNA with PI (red).

FIG. 21 depicts maintenance of normal mitochondrial dynamics in oocytesof aged females by CR. (A) Representative mitochondrial distribution inMII oocytes from 3M AL-fed, 12M AL-fed and 12M CR-AL-fed mice (stainingshown in red). (B) Incidence of abnormal mitochondrial aggregation inMII oocytes from 3M AL-fed, 12M AL-fed and 12M CR-AL-fed mice (mean±SEM,n=23-46 oocytes analyzed per group from 3 independent experiments using4-11 mice per group; *, P<0.05 vs. 3M AL-fed females). (C) CytoplasmicATP levels in individual MII oocytes from 3M AL-fed, 12M AL-fed and 12MCR-AL-fed mice (mean±SEM, n=38-145 total oocytes analyzed per group from5-7 independent experiments using a total of 5-21 mice per group; *,P<0.05 vs. 3M AL-fed females).

FIG. 22 depicts improvement in oocyte yield and quality in aging femalesresulting from the loss of peroxisome proliferator-activated receptor γcoactivator-1α (PGC-1α). (A) RT-PCR analysis of Pgc-1α and Pgc-1β mRNAlevels in isolated MII oocytes of 3M AL-fed wild-type (wt) mice, 12MAL-fed or CR-AL-fed wt mice, or 12M AL-fed or CR-AL-fed Pgc-1α-null mice(Actin, control gene for sample loading; Size, molecular size marker;Ov, adult ovary RNA used as a positive control; —RT, RT-PCR analysis ofovary RNA without reverse transcriptase as a negative control). (B-E)Effects of PGC-1α deficiency in AL-fed and CR-AL-fed females on oocyteyield following superovulation (B), meiotic spindle formation (C),chromosomal alignment on the metaphase plate (D), and mitochondrialdistribution (E) are shown. Legends for (D) and (E) are the same as (C).Data are the mean±SEM (n=20-117 oocytes analyzed per group for eachendpoint from 3 independent experiments using a total of 3-14 mice pergroup; *, P<0.05 vs. all other groups).

FIG. 23 depicts expression of PGC-1 is in oocytes. Immunohistochemicaldetection of PGC-1 (brown reaction product against blue hematoxylincounterstain) in young adult mouse ovaries. Insets show magnified imagesof typical positive oocytes.

FIG. 24 depicts diminished ovarian reserve with age in mice lackingPGC-1α. Number of non-atretic quiescent (primordial) and early growing(primary, preantral) immature follicles per ovary in 3M AL-fed, 12MAL-fed or 12M AL-CR-fed wild-type (wt) or PGC-1α-deficient (null) femalemice. Data are the mean±SEM (n=4-12 mice per group; *, P<0.05 vs. 3MAL-fed females of either genotype).

FIG. 25 depicts comparable levels of PGC-1 in ovaries of young and agedfemale mice. (A) Western blot analysis of endogenous PGC-1 proteinlevels in ovaries of young (3M) AL-fed, aged (12M) AL-fed, and aged(12M) CR-AL-fed females (samples prepared from 3 different mice areshown for each group). Pan-actin (ACTIN) was used as a loading control.(B) Examples of immunohistochemical detection of PGC-1 (brown reactionproduct against blue hematoxylin counterstain) in ovaries of the samefemales that were used to obtain samples for PGC-1 Western blotting (A).

FIG. 26 depicts the effects of dietary manipulation on body weight. Bodyweight of female mice just prior to initiation of the CR diet (3M), uponcompletion of the CR regimen (11M), and one month following theresumption of AL feeding (12M) are shown. Data shown are the mean±SEMfrom analysis of 5-23 mice per group (*, P<0.05 vs. 3M AL-fed females ineach respective group). JAX, C57BL/6 mice from Jackson Laboratories;NIA, C57BL/6 mice from the NIA; Pgc-1α, mutant mouse line obtained fromB. M. Spiegelman (Lin et al. Cell 2004 119: 121-135).

FIG. 27 depicts prevention of aging-related disruption of the femalereproductive cycle by CR. Proportion of aged (12M) AL-fed and CR-AL-fedfemales that exhibited a typical 4-5 day estrous cycle or atypicalestrous cycles lasting longer than 5 day. Data are from analysis of10-15 mice per group analyzed in parallel by daily vaginal smears over a30-day period.

FIG. 28 depicts mitochondrial DNA copy number (relative to nucleargenome). Mouse OSCs maintained in culture (Zou et al., Nat Cell Biol2009 11: 631-636) were exposed to the indicated test compounds for 24hours and then assessed for mitochondrial DNA (mtDNA) content.

FIG. 29 depicts mitochondrial membrane potential. Mouse OSCs maintainedin culture (Zou et al., Nat Cell Biol 2009 11: 631-636) were exposed tothe indicated test compounds for 24 hours and then assessed formitochondrial membrane potential (MMP).

FIG. 30 depicts mitochondrial DNA copy number (relative to the nucleargenome). Mouse OSCs maintained in culture (Zou et al., Nat Cell Biol2009 11: 631-636) were exposed to the indicated test compounds for 24hours and then assessed for mitochondrial DNA copy number.

FIG. 31 depicts ATP levels as a measure of mitochondrial activity. MouseOSCs maintained in culture (Zou et al., Nat Cell Biol 2009 11: 631-636)were exposed to the indicated test compounds for 24 hours and thenassessed for mitochondrial activity.

FIG. 32 depicts the percent increase in mitochondrial density asmeasured by the dye NAO. Mouse OSCs maintained in culture (Zou et al.,Nat Cell Biol 2009 11: 631-636) were exposed to the indicated testcompounds for 24 hours and then assessed for mitochondrial density.

FIG. 33 depicts mRNA levels of genes known to drive mitochondrialbiogenesis and energetics. Mouse OSCs maintained in culture (Zou et al.,Nat Cell Biol 2009 11: 631-636) were exposed to the indicated testcompounds for 24 hours and then assessed for expression levels of genesknown to drive mitochondrial biogenesis.

FIG. 34 depicts mRNA levels of genes encoding mitochondrial electrontransport chain components. Mouse OSCs maintained in culture (Zou etal., Nat Cell Biol 2009 11: 631-636) were exposed to the indicated testcompounds for 24 hours and then assessed for expression levels of genesencoding mitochondrial electron transport chain components.

FIG. 35 depicts levels of NAD+ (A) and NADH (B) in OSCs treated withmitochondrial enhancers. Mouse OSCs maintained in culture (Zou et al.,Nat Cell Biol 2009 11: 631-636) were exposed to the indicated testcompounds for 24 hours and then assessed for levels of NAD+ and NADH.

FIG. 36 depicts mitochondria following staining with mitotracker M7514and cell lysis. Human OSCs were incubated with M7514, and then lysed torelease the stained mitochondria using osmotic shock. The entirepopulation (mitochondria from lysed cells and residual unlysed stainedcells) was analyzed by FACS. The left panel shows mitochondria fromlysed cells, which are easily distinguishable from mitochondriacontained in residual unlysed cells based on size (forward scatter;FSC-A). Fluorescence intensity (FITC-A) revealed two distinctpopulations of mitochondria from lysed cells, one having high intensity(Mito MT high), and one having low intensity (Mito MT Low). Functionalmitochondria are known to have a greater uptake and retention of thestain, and thus fluoresce at a higher intensity.

FIG. 37 is a schematic diagram depicting pathways for the synthesis anddegradation of NAD⁺ in mammalian cells (Hassa, P. et al., Microbiol.Mol. Biol. Rev. September 2006 vol. 70 no. 3 789-829).

FIG. 38 shows the structures of exemplary soluble precursors to NAD⁺.These agents can be used to raise cellular NAD⁺ levels and boostcellular energetics in damaged and/or aged cells.

FIGS. 39A-39C are graphs showing that nicotinamide mononucleotides raisecellular NAD⁺ and NAD+/NADH in murine oogonial stem cells (OSCs).Oogonial stem cells were isolated from dissociated ovaries using a FACSbased sorting protocol to purify OSCs free of contaminating oocytes (seeExample 1). Cells were maintained in culture medium consisted of minimumessential medium α (MEMα), 10% FBS, 1 mM sodium pyruvate, 1 mMnon-essential amino acids, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol(Sigma Aldrich Corporation, St. Louis, Mo., USA), 10 ng/ml-1 LIF(Millipore), 1× N-2 MAX Media Supplement (R&D) 10 ng/ml EGF (Epidermalgrowth factor, Recombinant human; Gibco Division of ThermoFisherScientific, Waltham, Mass., USA), 40 ng/ml human GDNF (glial cellline-derived neurotrophic factor; R&D systems), 1 ng/ml human bFGF(basic fibroblast growth factor; Gibco Division of ThermofisherScientific, Waltham, Mass., USA).

FIG. 40 is a graph showing that nicotinamide mononucleotide increasesmitochondrial DNA content in murine OSCs. Total cellular DNA wasisolated from cells at the indicated time points using DNEASY® Blood &Tissue Kit (Qiagen, Venlo, The Netherlands) according to themanufacturer's instructions. Mt DNA copy number was quantified usingLIGHTCYCLER®480 SYBR® Green I Master (Roche Applied Science, Penzberg,Germany) using a LIGHTCYCLER®480 PCR machine (Roche Applied Science,Penzberg, Germany).

FIG. 41 is a graph showing that nicotinamide mononucleotide increasesspontaneous oocyte formation in cultured murine oogonial stem cells. Forassessment of spontaneous oocyte formation, each well of a 24- wellplate was seeded with 25,000 OSCs, and the number of oocytes formed andreleased into the medium per well was assessed the second day afterseeding as well as the designated time points after NMN treatment.

FIG. 42 is a graph showing that the NAD⁺ precursor NMN raises NAD⁺levels in vivo in young and old mice. Cardiac [NAD⁺] declines with ageand is reversed by NMN treatment (n=3; 200 mg.kg.d. I.P. for 1 week).

FIGS. 43A-D are graphs showing the restorative effects of an NAD⁺precursor (NMN) on mitochondrial function in vivo. The decline inmitochondrial function in skeletal muscle of 24-month old mice iscompletely reversed by NMN (nicotinamide mononucleotide) after only 1week of treatment (FIGS. 43A, B). NMN is delivered by intraperitoneal(I.P.) injection and raises NAD⁺ levels in brain, heart and skeletalmuscle ˜30-100%. NMN increases mitochondrial function in C2C12 cells ina SIRT1-dependent manner (FIGS. 43C, D). sh Ctl=scrambled shRNA, ShSIRT1=shRNA against SIRT1.

FIG. 44 is a bar graph showing the effects of apigenenin, luteolin andSRT-1720 on oocyte yield from aged female mice.

FIG. 45 is a bar graph showing the effects of apigenenin, luteolin andSRT-1720 on the percentage of mature, metaphase II oocytes retrievedfollowing superovulation as compared to aged female mice.

FIG. 46 is a bar graph showing that apigenenin, luteolin and SRT-1720improve the quality of oocytes in aged mice as compared to aged femalemice.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control.

The term “administration” or “administering” includes routes ofintroducing a compound(s) to a subject to perform their intendedfunction. Examples of routes of administration that can be used includeinjection (subcutaneous, intravenous, parenterally, intraperitoneally,intrathecal), oral, and transdermal. The pharmaceutical preparationsare, of course, given by forms suitable for each administration route.For example, these preparations are administered in tablets or capsuleform, by injection, or inhalation. Oral administration is preferred. Theinjection can be bolus or can be continuous infusion. Depending on theroute of administration, the compound can be coated with or disposed ina selected material to protect it from natural conditions which maydetrimentally effect its ability to perform its intended function.

The agent can be administered alone, or in conjunction with eitheranother agent as described above (e.g. another bioenergetic agent) orwith a pharmaceutically-acceptable carrier, or both. The compound can beadministered prior to the administration of the other agent,simultaneously with the agent, or after the administration of the agent.Furthermore, the compound can also be administered in a proform which isconverted into its active metabolite, or more active metabolite in vivo.

As used herein, the term “advanced maternal age” as it relates to humansrefers to a woman who is 34 years of age or older. As used herein, theterm “oocyte-related infertility” as it relates to humans refers to aninability to conceive after one year of unprotected intercourse which isnot caused by an anatomical abnormality (e.g., blocked oviduct) orpathological condition (e.g., uterine fibroids, severe endometriosis,Type II diabetes, polycystic ovarian disease)

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. The term alkyl further includesalkyl groups, which can further include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. Inpreferred embodiments, a straight chain or branched chain alkyl has 30or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain,C₃-C₃₀ for branched chain), preferably 26 or fewer, and more preferably20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atomsin their ring structure, and more preferably have 3, 4, 5, 6 or 7carbons in the ring structure.

Moreover, the term alkyl as used throughout the specification and claimsis intended to include both “unsubstituted alkyls” and “substitutedalkyls,” the latter of which refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six, and most preferablyfrom one to four carbon atoms in its backbone structure, which may bestraight or branched-chain. Examples of lower alkyl groups includemethyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl andso forth. In preferred embodiment, the term “lower alkyl” includes astraight chain alkyl having 4 or fewer carbon atoms in its backbone,e.g., C₁-C₄ alkyl.

The term “alkoxy,” as used herein, refers to an alkyl or a cycloalkylgroup which is linked to another moiety though an oxygen atom. Alkoxygroups can be optionally substituted with one or more substituents.

The terms “alkoxyalkyl,” “polyaminoalkyl” and “thioalkoxyalkyl” refer toalkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. For example, the invention contemplates cyano andpropargyl groups.

The term “aryl” refers to the radical of aryl groups, including 5- and6-membered single-ring aromatic groups that may include from zero tofour heteroatoms, for example, benzene, pyrrole, furan, thiophene,imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole,pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groupsalso include polycyclic fused aromatic groups such as naphthyl,quinolyl, indolyl, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles,”“heteroaryls” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “halogen” or “halo” designates —F, —Cl, —Br or —I.

The term “haloalkyl” is intended to include alkyl groups as definedabove that are mono-, di- or polysubstituted by halogen, e.g.,fluoromethyl and trifluoromethyl.

The term “hydroxyl” means —OH.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorus.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, andthe remainder ring atoms being carbon. Heteroaryl groups may beoptionally substituted with one or more substituents. Examples ofheteroaryl groups include, but are not limited to, pyridyl, furanyl,benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolylthiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl,isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl,imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl,benzothiadiazolyl, benzoxadiazolyl, and indolyl.

The term “heterocyclic” as used herein, refers to organic compounds thatcontain at least at least one atom other than carbon (e.g., S, O, N)within a ring structure. The ring structure in these organic compoundscan be either aromatic or non-aromatic. Some examples of heterocyclicmoeities include, are not limited to, pyridine, pyrimidine, pyrrolidine,furan, tetrahydrofuran, tetrahydrothiophene, and dioxane. “Bioenergeticagents,” as used herein, refer to agents that enhance mitochondrialnumbers, mitochondrial activity, cellular energy levels or cellularenergy-producing potential (bioenergetic status) in oocytes, OSCs and/orpreimplantation embryos prior to conducting and/or following methods ofin vitro fertilization, or following exposure of ovaries, oocytes, OSCsand/or preimplantation embryos in vivo. In particular, by enhancingmitochondrial numbers or activity, bioenergetic agents of the inventionimprove oocyte or OSC production and quality, for example, by preventingor decreasing aging-related increases in oocyte aneuploidy, chromosomalmisalignment on the metaphase plate, meiotic spindle abnormalities,and/or mitochondrial dysfunction (aggregation, impaired ATP production).Bioenergetic agents include Sirtl activators. Exemplary Sirtl activatorsare listed in Table 1 (below).

TABLE 1 Sirt1 Activators Compound: Resveratrol(3,5,4′-Trihydroxy-trans-stilbene) Butein(3,4,2′,4′-Tetrahydroxychalcone) Piceatannol(3,5,3′,4′-Tetrahydroxy-transstilbene) Isoliquiritigen(4,2′,4′-Trihydroxychalcone) Fisetin (3,7,3′,4′-Tetrahydroxyflavone)5,7,3′,4′,5′-Pentahydroxyflavone Luteolin(5,7,3′,4′-Tetrahydroxyflavone) 3,6,3′,4′-Tetrahydroxyflavone Quercetin(3,5,7,3′,4′-Pentahydroxyflavone) 7,3′,4′,5′-TetrahydroxyflavoneKaempferol (3,5,7,4′-Tetrahydroxyflavone) 6-Hydroxyapigenin(5,6,7,4′-Tetrahydroxyflavone; Scutellarein)3,4,2′,4′,6′-Pentahydroxychalcone Apigenin (5,7,4′-Trihydroxyflavone)Hinokitiol (b-Thujaplicin;2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1- one) Daidzein(7,4′-Dihydroxyisoflavone) Naringenin (5,7,4′-Trihydroxyflavanone)3,6,2′,4′-Tetrahydroxyflavone L-(+)-Ergothioneine((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-1Himidazole-4-ethanaminium inner salt)3,5,7,3′,4′-Pentahydroxyflavanone Deoxyrhapontin(3,5-Dihydroxy-4′-methoxystilbene 3-O-β-D-glucoside) Flavanone7,8,3′,4′-Tetrahydroxyflavone 7,4′-Dihydroxyflavone Caffeic Acid PhenylEster 3,6,2′,3′-Tetrahydroxyflavone 4′-Hydroxyflavone Pelargonidinchloride (3,5,7.4′-Tetrahydroxyflavylium chloride) 5,4′-Dihydroxyflavone(−)-Epicatechin (Hydroxy Sites: 3,5,7,3′,4′) 5,7-Dihydroxyflavonetrans-Stilbene Morin (3,5,7,2′,4′-Pentahydroxyflavone) Flavone(−)-Catechin (Hydroxy Sites: 3,5,7,3′,4′) Rhapontin(3,3′,5-Trihydroxy-4′-methoxystilbene 3-O-B-β-glucoside)(−)-Gallocatechin (Hydroxy Sites: 3,5,7,3′,4′,5′) Chalcone (+)-Catechin(Hydroxy Sites: 3,5,7,3′,4′) (+)-Epicatechin (Hydroxy Sites:3,5,7,3′,4′) MCI-186 (3-Methyl-1-phenyl-2-pyrazolin-5-one)5-Hydroxyflavone HBED(N,N′-Di-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid•HCl•H2O)cis-Stilbene Genistein (5,7,4′-Trihydroxyisoflavone) Ambroxol(trans-4-(2-Amino-3,5-dibromobenzylamino) cyclohexane•HCl) U-83836E((−)-2-((4-(2,6-di-1-Pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl)methyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol•2HCl)

CD38 inhibitors are also useful as bioenergetic agents. Exemplary CD38inhibitors are listed in Tables 2A and 2B (below).

TABLE 2A CD38 Inhibitors1-[(2-Acetoxyethoxy)methyl]-3-(aminocarbonyl)-pyridinium chloride1-[(2-Benzyloxyethoxy)methyl]-3-(aminocarbonyl)-pyridinium chloride1-{[2-(4-Methoxy-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-pyridiniumchloride1-{[2-(4-Phenoxy-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-pyridiniumchloride1-{[2-(4-Nitro-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-pyridiniumchloride1-{[2-(3-Trifluoromethyl-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-pyridiniumchloride1-{[2-(8′-Quinolyloxy)ethoxy]methyl}-3-(aminocarbonyl)-pyridiniumchloride 1,2-Dimethoxy-ethylene-bis-N,N′-3-(aminocarbonyl)-pyridiniumdichloride 1,4-Dimethoxy-butylene-bis-N,N′-3-(aminocarbonyl)-pyridiniumdichloride 1,4-Dimethoxy-butyne-bis-N,N′-3-(aminocarbonyl)-pyridiniumdichloride1,4-Dimethoxy-hexamethylene-bis-N,N′-3-(aminocarbonyl)-pyridiniumdichloride(E)-1-{[4-(8′-Quinolyloxy)but-2-enyloxy]methyl}-3-(aminocarbonyl)-pyridiniumchloride1-{[2-(4-Phenoxy-phenoxy)ethoxy]methyl}-6-(aminocarbonyl)-quinoliniumchloride1-{[2-(4-Phenoxy-phenoxy)ethoxy]methyl}-3-(aminocarbonyl)-4-amino-pyridiniumchloride

Additional CD38 inhibitors are listed in Table 2B.

Luteolinidin Kuromanin Luteolin Delphinidin Pelargonidin MalvidinQuercetagetinidin Peonidin Myricetin Cyanidin Diosmetinidin QuercetinRobinetin Petunidin Fisetinidin Quercetagetin rac-Taxifolin rac-CatechinPiceatannol Resveratrol Apigenin

Preferred bioenergetic agents for such uses include, but are not limitedto, soluble precursors to NAD⁺ (e.g., tryptophan, quinolinic acid,nicotinamide mononucleotide, nicotinamide riboside, and nicotinic acid),fisetin, quercetin, hydroxytyrosol (4-(2-Hydroxyethyl)-1,2-benzenediol),pyrroloquinoline quinone (PQQ), metformin, apigenin, luteolin,tyrphostin-8, berberine a CD38 inhibitor, and SRT-1720, a compound ofany one of formulas I-XV, and functional derivatives thereof.

DOI (2,5-dimethoxy-4-iodo-phenylisopropylamine) is a phenylalkylaminethat has been characterized as a 5-HT2-selective agonist.

Fisetin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxychromen-4-one) isdescribed, for example, by Herzig, Monatshefte für Chemie 1891 12:177-90; Gabor et al., Nature 1966 212 (5067): 1273; and Maher et al.,PLoS ONE 2011 6 (6): e21226, each of which is incorporated by reference.Quercetin is described, for example, by Bentz, The Journal of YoungInvestigators: Appalachian State University. [Online] Apr. 1, 2009,which is incorporated herein by reference.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is described, forexample, by Takaoka, Nippon Kagaku Kaishi 1939 60: 1090-1100; Hathway etal., Biochemical Journal 1959 72: 369-374; and Nonomuraet al., YakugakuZasshi 1963 83: 988-990, each of which is incorporated by reference.

Pyrroloquinoline quinone(4,5-Dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylicacid) is described, for example, by Hauge J Biol Chem 1964 239: 3630-9;Anthony et al., Biochem J 1967 104: 960-9; Salisbury et al., Nature 1979280: 843-4; Westerling et al., Biochem Biophys Res Commun 1979 87:719-24; Ameyama FEBS Lett 1981 130: 179-83, each of which isincorporated by reference.

Metformin (N,N-dimethylimidodicarbonimidic diamide) is described, forexample, by Werner. J Chem Soc, Transactions 1921 121: 1790-5; Shapiroet al., J Am Chem Soc. 1959 81: 2220-5; Patent FR 2322860 1975 French;and Pharmaceutical Manufacturing Encyclopedia (Sittig's PharmaceuticalManufacturing Encyclopedia). 3rd ed. Vol. 3. Norwich, N.Y.: WilliamAndrew; 2007, each of which is incorporated by reference.

Apigenin (5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) isdescribed, for example, by Merck Index, 11th Edition, 763, which isincorporated herein by reference.

Luteolin (2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone)_sdescribed, for example, by Mann Secondary Metabolism 1992 (2nd ed.).Oxford, UK: Oxford University Press. pp. 279-280; and Lopez-Lázaro MiniRev Med Chem 2009 9: 31-59.

Tyrphostin-8 (2[(4-hydroxyphenyl)methylidene]propanedinitrile) isdescribed, for example, by Martin; Biochem. Pharmacol. 1998 56: 483;Wolbring, et al.; J. Biol. Chem. 1994 269: 22470; Stanley, et al., J.Immunol. 1990 145: 2189, and Gazit, et al., J. Med. Chem. 1989 32: 2344.

Berberine(9,10-dimethoxy-5,6-dihydro[1,3]dioxolo[4,5-g]isoquino[3,2-a]isoquinolin-7-ium)is described, for example, by Dewick Medicinal Natural Products: ABiosynthetic Approach (3rd ed.). West Sussex, England: Wiley. 2009 p.357-358.

SRT-1720(N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide)is described, for example, by Milne et al., Nature 2007 450: 712-6.

“Oogonial stem cells” (OSCs), also known as female germline stem cells,are derived from postnatal sources and express markers including Vasa,Oct-4, Dazl, Stella and optionally an SSEA. OSCs are mitoticallycompetent (i.e., capable of mitosis) and do not express oocyte markersincluding growth/differentiation factor-9 (“GDF-9”) and zona pellucidaglycoproteins (e.g., zona pellucida glycoprotein-3, “ZP3”), or markersof meiotic recombination such as synaptonemal complex protein-3 (“SYCP3”or “SCP3”). OSCs can be obtained from the postnatal ovary. OSCs areknown in the art and are described in U.S. application Ser. No.11/131,114, filed on May 17, 2005 as Attorney Docket No. 51588-62054 andpublished as U.S. Patent Pub. No. 20060010508, the contents of which areincorporated herein by reference. OSCs are additionally described by Zouet al., Nat Cell Biol 2009 11: 631-636 and Pacchiarotti et al.Differentiation 2010 79: 159-170, the contents of which are incorporatedherein by reference. Preferably, the OSC of the invention is a humanOSC.

As used herein, the “progeny of an OSC” refers to all daughter cellsderived from OSCs of the invention, including progenitor cells anddifferentiated cells, which maintain oogenic potential (i.e., theability to form an oocyte). Preferably, the OSC progeny of the inventionis a human OSC progeny.

OSCs may additionally be obtained from the bone marrow, peripheral bloodor umbilical cord blood. Bone marrow derived OSCs of the invention canalso circulate throughout the body and most preferably can be localizedin bone marrow, peripheral blood and ovary. Bone marrow derived OSCsexpress markers including Oct 4, Vasa, Daz1, Stella, Fragilis, andoptionally Nobox, Kit and Sca-1. Bone marrow derived OSCs aremitotically competent (i.e., capable of mitosis) and do not expressGDF-9, zona pellucida proteins (e.g., ZP3) or SCP3. For additionaldetails on bone marrow-derived OSCs, see, U.S. application Ser. No.11/131,153, filed on May 17, 2005 as Attorney Docket No. 51588-62060 andpublished as U.S. Patent Pub. No. 20060010509, the contents of which areincorporated herein by reference for their description of OSCs in thebone marrow. For additional details on peripheral blood and umbilicalcord blood derived OSCs, see U.S. application Ser. No. 11/131,152, filedon May 17, 2005 as Attorney Docket No. 51588-62065 and published as U.S.Patent Pub. No. 20060015961, the contents of which are incorporatedherein by reference for their description of OSCs in the peripheralblood.

Oct-4, also referred to as POU domain class 5 transcription factor 1 orPou5f1, is a gene expressed in female germline stem cells and theirprogenitor cells. The Oct-4 gene encodes a transcription factor that isinvolved in the establishment of the mammalian germline and plays asignificant role in early germ cell specification (reviewed in Scholer,Trends Genet. 1991 7(10): 323-329). In the developing mammalian embryo,Oct-4 is down-regulated during the differentiation of the epiblast,eventually becoming confined to the germ cell lineage. In the germline,Oct-4 expression is regulated separately from epiblast expression.Expression of Oct-4 is a phenotypic marker of totipotency (Yeom et al.,Development 1996 122: 881-888).

Stella, also commonly referred to as developmental pluripotencyassociated 3 or Dppa3, is a gene expressed in female germline stem cellsand their progenitor cells. Stella is a novel gene specificallyexpressed in primordial germ cells and their descendants, includingoocytes (Bortvin et al., BMC Developmental Biology 2004 4(2): 1-5).Stella encodes a protein with a SAP-like domain and a splicing factormotif-like structure. Embryos deficient in Stella expression arecompromised in preimplantation development and rarely reach theblastocyst stage. Thus, Stella is a maternal factor implicated in earlyembryogenesis.

Dazl is a gene expressed in female germline stem cells and theirprogenitor cells. The autosomal gene Dazl is a member of a family ofgenes that contain a consensus RNA binding domain and are expressed ingerm cells. Loss of expression of an intact Dazl protein in mice isassociated with failure of germ cells to complete meiotic prophase.Specifically, in female mice null for Dazl, loss of germ cells occursduring fetal life at a time coincident with progression of germ cellsthrough meiotic prophase. In male mice null for Dazl, germ cells wereunable to progress beyond the leptotene stage of meiotic prophase I.Thus, in the absence of Dazl, progression through meiotic prophase isinterrupted (Saunders et al., Reproduction 2003 126: 589-597).

Vasa, also referred to as DEAD box polypeptide 4 or Ddx4 (Asp Glu AlaAsp—SEQ ID NO: 1), is a gene expressed in female germline stem cells andtheir progenitor cells. Vasa is a component of the germplasm thatencodes a DEAD-family ATP-dependent RNA helicase (Liang et al.,Development 1994 120: 1201-1211; Lasko et al., Nature 1988 335:611-167). The molecular function of Vasa is directed to binding targetmRNAs involved in germ cell establishment (e.g., Oskar and Nanos),oogenesis, (e.g., Gruken), and translation onset (Gavis et al.,Development 1996 110: 521-528). Vasa is required for pole cell formationand is exclusively restricted to the germ cell lineage throughout thedevelopment. Thus, Vasa is a molecular marker for the germ cell lineagein most animal species (Toshiaki et al., Cell Structure and Function2001 26: 131-136).

Stage-Specific Embryonic Antigens are optionally expressed in femalegermline stem cells and expressed in female germline stem cellprogenitors of the invention. Stage-Specific Embryonic Antigen-1(SSEA-1) is a cell surface embryonic antigen whose functions areassociated with cell adhesion, migration and differentiation. Duringhypoblast formation, SSEA-1 positive cells can be identified in theblastocoel and hypoblast and later in the germinal crescent. SSEA-1functions in the early germ cell and neural cell development. (D′Costaet al., Intl. Dev. Biol. 1999 43(4): 349-356; Henderson et al., StemCells 2002 20: 329-337). In specific embodiments, expression of SSEAs infemale germline stem cells may arise as the cells differentiate. SSEAsuseful in the invention include SSEA-1, -2, -3, and -4.

The term “autologous” as used herein refers to biological compositionsobtained from the same subject. In one embodiment, the biologicalcomposition includes OSCs, OSC-derived compositions and oocytes.Accordingly, in conducting methods of the invention, the female germcell cytoplasm or mitochondria used for transfer and the recipientoocyte into which the aforementioned compositions are transferred areobtained from the same subject.

The term “increase” as used herein generally means an increase of atleast 5%, for example an increase by at least about 10%, or at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increase(i.e. substantially above levels of detection), or any increase between5-100% as compared to a reference level, as that term is defined herein,and as determined by a method that achieves statistical significance(p<0.05).

The term “isolated” as used herein refers to an OSC, mitochondrion orcomposition derived from an OSC (e.g., cytoplasm, mitochondrialpreparation), which has been physically separated or removed from itsnatural biological environment. An isolated OSC, mitochondrion orcomposition need not be purified. The biological sample can include, forexample, bone marrow, peripheral blood, umbilical cord blood, ovary orspleen or cells obtained from bone marrow, peripheral blood, ovary orspleen. Preferably, the composition comprises at least 50%, 75%, 85%,90%, 95% or 100% of the cell type or organelle of interest relative toother cell types or organelles.

As used herein, the term “low ovarian reserve” as it relates to humansrefers to a woman who exhibits a circulating Follicle StimulatingHormone (FSH) level greater than 15 miu/ml in a “day 3 FSH test,” asdescribed in Scott et al., Fertility and Sterility, 1989 51: 651-4, or acirculating Anti-Mullerian Hormone (AMH) level less than 0.6 ng/ml, oran antral follicle count less than 7 as measured by ultrasound.

The term “exogenous” as used herein refers to transferred cellularmaterial (e.g., mitochondria) that is removed from one cell andtransferred into another cell. Preferably, the cells and transferredmaterials are autologous. For example, OSC derived mitochondria thathave been transferred into an oocyte, even if both are derived from thesame subject, would be exogenous.

The term “prodrug” includes compounds with moieties which can bemetabolized in vivo. Generally, the prodrugs are metabolized in vivo byesterases or by other mechanisms to active drugs. Examples of prodrugsand their uses are well known in the art (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19). The prodrugs can beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form or hydroxyl with a suitable esterifying agent. Hydroxyl groupscan be converted into esters via treatment with a carboxylic acid.Examples of prodrug moieties include substituted and unsubstituted,branch or unbranched lower alkyl ester moieties, (e.g., propionoic acidesters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters(e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g.,acetyloxymethyl ester), acyloxy lower alkyl esters (e.g.,pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkylesters (e.g., benzyl ester), substituted (e.g., with methyl, halo, ormethoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferredprodrug moieties are propionoic acid esters and acyl esters. Prodrugswhich are converted to active forms through other mechanisms in vivo arealso included.

Compounds that increase the activity of sirtuins, e.g., SIRT1, arereferred to as “SIRT1 activators.” Exemplary compounds are listed inTables 1, 2A, and 2B, and are described, e.g., in WO 05/002672, WO05/002555, US 20050136537, US 20060025337, WO 2005/065667 and WO2007/084162, and include polyphenols, e.g. plant polyphenols.

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease of at least 5%, for example a decrease by at leastabout 10%, or at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% decrease (i.e. substantially absent or below levels ofdetection), or any decrease between 5-100% as compared to a referencelevel, as that term is defined herein, and as determined by a methodthat achieves statistical significance (p<0.05).

A “subject” is a vertebrate, including any member of the class mammalia,including humans, domestic and farm animals, and zoo, sports or petanimals, such as mouse, rabbit, pig, sheep, goat, cattle and higherprimates.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “ includes,” “including,” and the like; “consistingessentially of or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease by a statistically significant amount. However, foravoidance of doubt, “reduced” means a decrease by at least 5% ascompared to a reference level, for example a decrease by at least about10%, or at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga 100% decrease (i.e. substantially absent or below levels ofdetection), or any decrease between 5-100% as compared to a referencelevel, as that term is defined herein.

The term “increase” as used herein generally means an increase by astatistically significant amount. However, for avoidance of doubt,“increase” means an increase by at least 5% as compared to a referencelevel, for example an increase by at least about 10%, or at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% or up to and including a 100% increase (i.e.significantly above levels of detection), or any increase between10-100% as compared to a reference level, as that term is definedherein.

As used herein, the term “standard” or “reference” refers to a measuredbiological parameter including but not limited to defects such asaneuploidy, mutation, chromosomal misalignment, meiotic spindleabnormalities, and/or mitochondrial dysfunction (aggregation, impairedATP production), or the reduction or elimination of such defects, in aknown sample against which another sample is compared; alternatively, astandard can simply be a reference number that represents an amount ofthe measured biological parameter that defines a baseline forcomparison. The reference number can be derived from either a sampletaken from an individual, or a plurality of individuals or cellsobtained therefrom (e.g., oocytes, OSCs). That is, the “standard” doesnot need to be a sample that is tested, but can be an accepted referencenumber or value. A series of standards can be developed that take intoaccount an individual's status, e.g., with respect to age, gender,weight, height, ethnic background etc. A standard level can be obtainedfor example from a known sample from a different individual (e.g., notthe individual being tested). A known sample can also be obtained bypooling samples from a plurality of individuals (or cells obtainedtherefrom) to produce a standard over an averaged population.Additionally, a standard can be synthesized such that a series ofstandards are used to quantify the biological parameter in anindividual's sample. A sample from the individual to be tested can beobtained at an earlier time point (presumably prior to the onset oftreatment) and serve as a standard or reference compared to a sampletaken from the same individual after the onset of treatment. In suchinstances, the standard can provide a measure of the efficacy oftreatment.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Other definitions appear in context throughout this disclosure.

Compositions and Methods of the Invention Bioenergetic Agents for Use inthe Invention

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula I or acompound of formula II:

wherein

is an aryl heterocycle diradical;

is heteroaryl;

X is halo;

R¹ is hydroxy, alkoxy, or amino; and

R² is hydroxy, alkoxy, or amino.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is a diradical of azaindole, benzo(b)thiene, benzimidazole, benzofuran,benzoxazole, benzothiazole, benzothiadiazole, benzotriazole,benzoxadiazole, furan, imidazole, imidazopyridine, indole, indoline,indazole, isoindoline, isoxazole, isothiazole, isoquinoline, oxadiazole,oxazole, purine, pyran, pyrazine, pyrazole, pyridine, pyrimidine,pyrrole, pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine, quinoline,quinazoline, triazole, thiazole, thiobenzene, tetrahydroindole,tetrazole, thiadiazole, thiophene, thiomorpholine, or triazole.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is a diradical of furan, imidazole, isoxazole, isothiazole, oxadiazole,oxazole, pyrrole, triazole, thiazole, tetrazole, thiadiazole, thiophene,or triazole.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is a diradical of imidazole.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl,indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl,isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl,pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, or triazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl,pyrrolyl, triazolyl, thiazolyl, tetrazolyl, thiadiazolyl, thienyl, ortriazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is imidazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein X is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is alkoxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is methoxy.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula III:

wherein, independently for each occurrence,

is heteroaryl;

X is halo;

R² is hydroxy, alkoxy, or amino; and

Y is —O— or —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl,indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl,isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl,pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, or triazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl,pyrrolyl, triazolyl, thiazolyl, tetrazolyl, thiadiazolyl, thienyl, ortriazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is imidazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein X is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is alkoxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is methoxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y is —O—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula IV:

wherein, independently for each occurrence,

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R¹ is hydroxy, alkoxy, or amino; and

Y¹ is —S—, —O—, or —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein one instance of R is hydroxy. In certain embodiments,the invention relates to any one of the aforementioned compositions,oocytes, or methods, wherein one instance of R is hydroxy; and theremaining instances of R are —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y¹ is —O—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula V:wherein, independently for each occurrence,

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino; and

R¹ is hydroxy, alkoxy, or amino.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, cells (e.g., OSCs, oocytes), or methods,wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula VI:

wherein, independently for each occurrence,

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R1 is hydroxy, alkoxy, or amino;

R³ is —H, cyano, —CO₂R⁴, or —C(O)N(R⁴)₂; and

R⁴ is —H or alkyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein at least one instance of R³ is cyano. In certainembodiments, the invention relates to any one of the aforementionedcompositions, tissues, cells (e.g., OSCs, oocytes), or methods, whereinat least two instances of R³ are cyano.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula VII:

wherein, independently for each occurrence,

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino; and

R¹ is hydroxy, alkoxy, or amino.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is compound of formula VIII:

wherein, independently for each occurrence,

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

X is halo;

Y¹ is —O—, —S—, or —NH—; and

Y² is ═N— or ═CR—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein X is chloro.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y¹ is —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y² is ═N—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula IX:

wherein, independently for each occurrence,

is a five-membered, unsaturated heterocycle diradical;

is heteroaryl;

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R¹ is hydroxy, alkoxy, or amino; and

Y² is ═N— or ═CR—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl,indolinyl, indazolyl, isoindolinyl, isoxazolyl, iothiazolyl,isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl,pyrazolyl, pyrdinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, or triazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein one instance of Y² is ═N—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula X:

wherein, independently for each occurrence,

is heteroaryl;

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

Y¹ is —S—, —O—, or —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl,indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl,isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl,pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, or triazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y¹ is —NH—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula XI:

wherein, independently for each occurrence,

is an aryl heterocycle diradical;

is heteroaryl;

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R¹ is hydroxy, alkoxy, or amino; and

Y² is ═N— or ═CR—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein

is azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl,indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl,isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl,pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, or triazolyl.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein one instance of Y² is ═N—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula XII:

wherein, independently for each occurrence,

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino; and

Y¹ is —S—, —O—, or —NH—.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein at least one instance of R is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein at least one instance of Y¹ is —S—.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula XIII:

wherein, independently for each occurrence,

is aryl or heteroaryl;

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R¹ is hydroxy, alkoxy, or amino;

R² is hydroxy, alkoxy, or amino;

R³ is —H, cyano, —CO₂R⁴, or —C(O)N(R⁴)₂;

X is halo;

Y³ is a bond, —C(O)-d, —C(O)NH-d, —NH—C(O)-d, or —C(O)NH—CH₂-d; and d isa bond to

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is alkoxy. In certain embodiments, the inventionrelates to any one of the aforementioned compositions, oocytes, ormethods, wherein R² is methoxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein one instance of R³ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R³ is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein X is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y³ is a bond. In certain embodiments, the inventionrelates to any one of the aforementioned compositions (e.g., OSCs,oocytes), or methods, wherein Y³ is —C(O)-d. In certain embodiments, theinvention relates to any one of the aforementioned compositions,tissues, cells (e.g., OSCs, oocytes), or methods, wherein Y³ is—C(O)NH-d. In certain embodiments, the invention relates to any one ofthe aforementioned compositions, tissues, cells (e.g., OSCs, oocytes),or methods, wherein Y³ is —NH— C(O)-d. In certain embodiments, theinvention relates to any one of the aforementioned compositions,tissues, cells (e.g., OSCs, oocytes), or methods, wherein Y³ is—C(O)NH—CH₂-d.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula XIV:

wherein, independently for each occurrence,

is a five-membered heterocycle radical;R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R¹ is hydroxy, alkoxy, or amino;

R² is hydroxy, alkoxy, or amino; and

X is halo.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is alkoxy. In certain embodiments, the inventionrelates to any one of the aforementioned compositions, oocytes, ormethods, wherein R² is methoxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein X is bromo.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is a compound of formula XV:

wherein, independently for each occurrence,

is aryl or heteroaryl;

R is —H, halo, aryl, nitro, alkyl, hydroxy, alkoxy, or amino;

R¹ is hydroxy, alkoxy, or amino;

R² is hydroxy, alkoxy, or amino;

R³ is —H, cyano, —CO₂R⁴, or —C(O)N(R⁴)₂;

X is halo;

Y³ is a bond, —C(O)-d, —C(O)NH-d, —NH—C(O)-d, or —C(O)NH—CH₂-d; and

d is a bond to

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R is —H

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R¹ is hydroxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R² is alkoxy. In certain embodiments, the inventionrelates to any one of the aforementioned compositions, oocytes, ormethods, wherein R² is methoxy.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein one instance of R³ is cyano.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein R³ is —H.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein X is bromo.

In certain embodiments, the invention relates to any one of theaforementioned compositions, tissues, cells (e.g., OSCs, oocytes), ormethods, wherein Y³ is a bond. In certain embodiments, the inventionrelates to any one of the aforementioned compositions, oocytes, ormethods, wherein Y³ is —C(O)-d. In certain embodiments, the inventionrelates to any one of the aforementioned compositions, tissues, cells(e.g., OSCs, oocytes), or methods, wherein Y³ is —C(O)NH-d. In certainembodiments, the invention relates to any one of the aforementionedcompositions, tissues, cells (e.g., OSCs, oocytes), or methods, whereinY³ is —NH—C(O)-d. In certain embodiments, the invention relates to anyone of the aforementioned compositions, tissues, cells (e.g., OSCs,oocytes), or methods, wherein Y³ is —C(O)NH—CH₂-d.

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is selected from the groupconsisting of

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is selected from the groupconsisting of

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is selected from the groupconsisting of

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is selected from the groupconsisting of

In certain embodiments, the invention relates to any one of thecompositions, tissues, cells (e.g., OSCs, oocytes), or methods describedherein, wherein the bioenergetic agent is selected from the groupconsisting of a-lineolic acid, lineolic acid, stearic acid, elaidicacid, arachidonic acid, oleic acid, and palmitoleic acid.

In alternate embodiments, one or more of the following bioenergeticagents is specifically excluded from the methods of the invention:tryptophan, quinolinic acid, nicotinamide mononucleotide, nicotinamideriboside, nicotinic acid, fisetin, quercetin, hydroxytyrosol,pyrroloquinoline quinone (PQQ), metformin, apigenin, luteolin,tyrphostin-8, berberine, SRT-1720, and

Pharmaceutically acceptable salts and prodrugs of the bioenergeticagents described herein may also be used.

Isolation of Oocytes and OSCs

Standard methods for the isolation of oocytes from human subjects usingprocedures such as transvaginal ultrasound guided oocyte retrieval arewell known in the art. See Fabbri et al., Hum Reprod 2001 16: 411-416.

Prior to isolation of OSCs, adult ovarian cortical tissue can beobtained using a minor laparoscopic procedure known in the art tocollect a small (e.g., 3×3×1 mm) ovarian biopsy, which is then processedfor OSC isolation. See Gook et al., Hum Reprod 2004 20: 72-78. Isolationof human OSCs from adult ovarian cortical tissue can be performed asdescribed in Example 1, FIG. 1 or as previously described. See, forexample, paragraph 0116 of U.S. Patent Pub. No. 20060010508, filed asU.S. application Ser. No. 11/131,114 on May 17, 2005 and Zou et al., NatCell Biol 2009 11: 631-636. OSCs can also be obtained from non-ovariansources, such as bone marrow or peripheral blood. Bone marrow andperipheral blood derived OSCs can be isolated by standard means known inthe art for the separation of stem cells from, for example, the marrowor blood (e.g., cell sorting). Optionally, the isolation protocolincludes generation of a kit+/lin-fraction that is depleted ofhematopoietic cells. Additional selection means based on the uniqueprofile of gene expression (e.g., Vasa, Oct-4, Dazl, Stella, Fragilis)can be employed to further purify populations of cells to an extentwhere they become substantially free of the biological sample from whichthey were obtained (e.g. bone marrow, peripheral blood, umbilical cordblood). For example, the methods described in Example 1, FIG. 1 havebeen applied to a mononuclear fraction of blood cells and bone marrowcells to obtain the purified OSCs from non-ovarian sources. Briefly,cells were incubated with a rabbit anti-VASA antibody (COOH-Antibody577-716) for 20 minutes (ab13840; Abcam, Cambridge, Mass., USA), washed,and incubated with goat anti-rabbit IgG conjugated to allophcocyanin(APC) for 20 minutes, and washed again. Labeled cells in the eluate wereisolated by fluorescence-activated cell sorting (FACS) using a FACSARIAII® cytometer (BD Biosciences, San Jose, Calif., Jose; provided byHarvard Stem Cell Institute, Boston, Mass.), gated against negative(unstained and no primary antibody) controls. Propidium iodide was addedto the cell suspension just prior to sorting for dead cell exclusion.Results obtained using cell surface expression of Vasa to isolate OSCsfrom non-ovarian sources are provided in FIGS. 14 and 15, where theFACS—based germ cell purification of bone marrow and peripheral bloodpreparations from adult female mice during estrus of the femalereproductive cycle is shown. Other antibodies for use in isolationmethods include those described in U.S. Pat. Nos. 7,884,193, 7,226,994and 6,875,854, the contents of which are incorporated herein byreference.

Preparation of Ooctye and OSC Derived Compositions and Methods ofTransfer

Methods for the preparation and transfer of purified mitochondria areknown in the art and can be carried out as previously described. See,for example, Perez et al., Cell Death Differ 2007 14: 524-533 and Perezet al., Nature 2000 , 403: 500-1, the contents of which are expresslyincorporated herein by reference. Briefly, OSCs and/or oocytes can beisolated and cultured as described above. Optionally, OSCs and/oroocytes can be isolated and cultured in the presence of one or morebioenegetic agents prior to mitochondrial extraction or preparation. Toobtain mitochondria from OSCs, OSC progeny and/or oocytes, 2 ml ofmitochondrial lysis buffer (0.3 M sucrose, 1 mM EDTA, 5 mM MOPS, 5 mMKH2PO4, 0.1% BSA) is added to each plate, and the cells are removedusing a cell scraper if necessary. The cell suspension is transferredinto a small glass tissue bouncer and homogenized until smooth(approximately 10 up-and-down strokes), and the lysate is centrifuged at600× g for 30 minutes at 4° C. The supernatant is removed and spun at10,000× g for 12 minutes at 4° C., and the resulting crude mitochondrialpellet is resuspended in 0.2 ml of 0.25 M sucrose. This sample is thenlayered over a 25-60% Percoll density gradient diluted with 0.25 Msucrose and centrifuged at 40,000× g for 20 minutes at 17° C. Theinterface band is extracted from the gradient and washed in 2 volumes of0.25 M sucrose before a final centrifugation at 14,000× g for 10 min at4° C. to yield a mitochondrial pellet.

The mitochondrial pellet can also be prepared as described Frezza et al.Nature Protocols 2007 2: 287-295, the contents of which are incorporatedherein by reference. In specific embodiments of the invention, the total0SC-derived mitochondrial population in a tissue, cell, lysed cell, orfraction thereof can be isolated, characterized and/or enumerated usinga FACS-based method with a fluorescent probe that specifically binds tomitochondria in a mitochondrial membrane potential (MMP)-independentmanner. Fluorescent probes that specifically bind to mitochondria in aMMP-independent manner include, but are not limited to, accumulationdependent probes (e.g., JC-1 (red spectrum; INVITROGEN® T3168),MitoTracker Deep Red FM (INVITROGEN® M22426) and JC-1 (green spectrum;INVITROGEN® T3168)). Functional (e.g., respiring) mitochondria can besorted and collected, preferably with exclusion of residual unlysedcells and non-functional mitochondria, based on size and fluorescenceintensity using mitochondrial tracking probes that indicatemitochondrial mass including, but not limited to, non-oxidationdependent probes (e.g., MitoTracker Green FM (INVITROGEN® M7514)).Details of an exemplary protocol for conducting FACS with anon-oxidation dependent probe are provided below in Example 10.Optionally, the FACS-based method can also be employed to selectivelyyield a pure population of functional (e.g., respiring) mitochondriausing a mitochondrial membrane fluorescent probe that specifically bindsto mitochondria in a MMP-dependent manner. Fluorescent probes thatspecifically bind to mitochondria in a MMP-dependent manner include, butare not limited to, reduced oxidative state mitotracker probes (e.g.,MitoTracker Red CM-H2XRos (Invitrogen M7513) and MitoTracker OrangeCM-H2TMRos (INVITROGEN® M7511). Furthermore, dual-labeling usingMMP-dependent and MMP-independent probes can be conducted to quantitatethe ratio of functional to total mitochondria in a tissue, cell, lysedcell or fraction derived thereof. When using probes for differentialscreening based on MMP, spectral color is the major determining factorto designate functional mitochondria, and forward scatter can be used todistinguish the fluorescent mitochondria released from lysed cells fromthose still contained in residual unlysed cells.

Mitochondrial pellets can also be prepared as described by Taylor etal., Nat Biotechnol. 2003 Mar; 21(3): 239-40; Hanson et al.,Electrophoresis. 2001 Mar; 22(5): 950-9; and Hanson et al., J Biol Chem.2001 May 11; 276(19): 16296-301. In specific embodiments of theinvention, the total OSC-derived mitochondrial population in a tissue,cell, lysed cell, or fraction thereof can be isolated, characterizedand/or enumerated using a differential centrifugation method asdescribed herein at Example 11 or using a sucrose gradient separationprocedure as described herein at Example 12.

Following isolation, assessment of mitochondrial DNA (mtDNA) integrity(e.g., mutations and deletions) can be conducted according to methodsknown in the art (Duran et al., Fertility and Sterility 2011 96(2):384-388; Aral et al., Genetics and Molecular Biology 2010 33: 1-4; Chanet al., Molecular Human Reproduction 2005 11(12): 843-846; Chen et al.,BMC Medical Genetics 2011 12: 8). Populations of mitochondria sortedaccording to functional parameters (e.g., MMP dependent/active orMMP-independent/active plus inactive) or mitochondria from lesspreferred OSC sources, including samples of limited size, can be now beobtained according to the methods of the invention.

Optionally, one or more bioenegetic agents can be added to themitochondrial preparation prior to mitochondrial extraction from cells,mitochondrial isolation from cell extracts or mitochondrial injection.Microinjection needles and holding pipettes can be made using a Sutterpuller (Sutter Instruments, Novato, Calif., USA) and a De FonbruneMicroforge (EB Sciences, East Granby, Conn., USA). The microinjectionneedles have inner diameters of 5 μm with blunt tips. The material to beinjected is aspirated into the needle by negative suction. Between about1×10³-to about 5×10⁴ mitochondria from OSCs or their progeny can beinjected (e.g., about 1, 2, 3, 4, 5, 6, 7, 8 to 9×10³; about 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 to about 5×10⁴mitochondria). Thematerial (e.g., mitochondrial suspension) in sucrose (e.g., 5-7 plcontaining approximately 1×10³-5×10⁴ mitochondria from OSCs or theirprogeny) can be injected into oocytes using a Piezo micromanipulator.Oocytes that survive the microinjection procedure are transferred forculture and optionally, assessment or cryopreservation prior to in vitrofertilization or intrauterine insemination. Optionally, mitochondrialsuspensions can be co-injected with a single sperm during in vitrofertilization, in a process referred to as intracytoplasmic sperminjection (ICSI). Optionally, oocytes can be cultured in the presence ofone or more bioenegetic agents prior to cryopreservation, in vitrofertilization or intrauterine insemination. Methods of oocytecryopreservation are well known in the art. For details, see Porcu etal., Mol Cell Endocrinol 2000 169: 33-37; Mandelbaum, Hum Reprod 200015: 43-47; Fabbri et al., Mol Cell Endocrinol 2000 169: 39-42, thecontents of which are incorporated herein by reference.

Methods for the preparation and transfer of nuclear-free cytoplasmicfractions are known in the art and can be carried out as previouslydescribed. See, for example, Cohen et al., Mol Hum Reprod 1998 4:269-280, the contents of which are incorporated herein by reference.Briefly, approximately 4 hours after egg retrieval, recipient eggs areexposed to 0.1% hyaluronidase, and mature eggs are selected forinjection. All corona cells are removed with fine bore pipettes.Ooplasmic transfer can be performed by electrofusion of OSC ooplast withintact MII oocytes. After exposure to 0.1% hyaluronidase zonae areopened mechanically using a microspear. OSCs and/or oocytes are exposedto hHTF medium containing cytochalosin B (CCB; Sigma AldrichCorporation, St Louis, Mo., USA) for 10 min at 37° C. Partitioning ofhuman MII oocytes involves variable cytochalasin B concentrationdepending on their sensitivity (˜2.5 mg/ml). Ooplasts of various sizesare separated from OSCs and/or oocytes by withdrawing a portion ofooplasm enclosed in the plasma membrane. Ooplasts can optionally becombined with bioenergetic agents. Alignment and electrofusion in amannitol solution is performed after insertion of the ooplast into theperivitelline space of the recipient egg from which the polar body wasremoved. This can be done with a wide-bored polished microtool ˜30-40 μmin diameter. The ooplast is sucked into the microtool and released oncethe tool is placed deeply into the perivitelline space. Oocytes thatsurvive the electrofusion procedure are transferred for culture andoptionally, assessment or cryopreservation prior to in vitrofertilization or intrauterine insemination. Optionally, oocytes can becultured in the presence of one or more bioenegetic agents prior tocryopreservation in vitro fertilization or intrauterine insemination.

Alternatively, conventional intracytoplasmic sperm injection (ICSI)methods can be employed in connection with the transfer of nuclear-freecytoplasmic fractions or isolated mitochondria, either with or withoutbioenegetic agents. See, for example, Cohen et al., Mol Hum Reprod 19984: 269-280, the contents of which are incorporated herein by reference.As one example, the zonae of the recipient eggs are opened mechanicallyover the polar body area using a microspear. The polar body is removedafter re-positioning the oocyte on the holding pipette in such a waythat the zona can be dissected using the closed microspear. The sameposition is used to insert the ooplast ˜90° left of the area, which hadcontained the polar body. The zona is closed tight using the same tool.Electrofused cells are washed and incubated in HTF for 40-90 min priorto ICSI. Spermatozoa are immobilized in 10% polyvinylpyrrolidone (PVP)for ICSI. The procedure is performed in HTF while the short side of theaperture is at approximately 3 o′clock. The ICSI tool is moved throughthe artificial gap in order to avoid extrusion of ooplasm uponindentation of the zona during standard ICSI. Zygotes can be cultured inthe presence of one or more bioenegetic agents prior to uterinetransfer.

Standard methods of in vitro fertilization are well known in the art.Couples are generally first evaluated to diagnose their particularinfertility problem(s). These may range from unexplained infertility ofboth partners to severe problems of the female (e.g., endometriosisresulting in nonpatent oviducts with irregular menstrual cycles orpolycystic ovarian disease) or the male (e.g., low sperm count withmorphological abnormalities, or an inability to ejaculate normally aswith spinal cord lesions, retrograde ejaculation, or reversedvasectomy). The results of these evaluations also determine the specificprocedure to be performed for each couple.

Procedures often begin with the administration of a drug todown-regulate the hypothalamic/pituitary system (gonadotropin-releasinghormone or GnRH agonist). This process decreases serum concentrations ofthe gonadotropins, and developing ovarian follicles degenerate, therebyproviding a set of new follicles at earlier stages of development. Thispermits more precise control of the maturation of these new follicles byadministration of exogenous gonadotropins in the absence of influencesby the hypothalamic pituitary axis. The progress of maturation and thenumber of growing follicles (usually four to ten stimulated per ovary)are monitored by daily observations using ultrasound and serum estradioldeterminations. When the follicles attain preovulatory size (18-21 mm)and estradiol concentrations continue to rise linearly, the ovulatoryresponse is initiated by exogenous administration of human chorionicgonadotropin (hCG).

Prior to the transplantation procedure, individual oocytes can beevaluated morphologically and transferred to a petri dish containingculture media and heat-inactivated serum and optionally, oocytes can becultured in the presence of one or more bioenegetic agents. A semensample is provided by the male partner and processed using a “swim up”procedure, whereby the most active, motile sperm will be obtained forinsemination. If the female's oviducts are present, a procedure calledGIFT (gamete intrafallopian transfer) can be performed at this time. Bythis approach, oocyte-cumulus complexes surrounded by sperm are placeddirectly into the oviducts by laparoscopy, wither with or withoutbioenergetic agents. This procedure best simulates the normal sequencesof events and permits fertilization to occur within the oviducts. Notsurprisingly, GIFT has the highest success rate with 22% of the 3,750patients undergoing ova retrieval in 1990 having a live delivery. Analternative procedure ZIFT (zygote intrafallopian transfer) permits theselection of preimplantation embryos derived from in vitro fertilizedzygotes to be transferred to oviducts the day following ova retrieval,either with or without bioenergetic agents. Extra zygotes and/orpreimplantation embryos can be cryopreserved at this time for futuretransfer or for donation to couples without female gametes. Mostpatients having more serious infertility problems, however, will requirean additional one to two days incubation in culture so thatpreimplantation embryos in the early cleavage states can be selected fortransfer to the uterus. This IVF-UT (in vitro fertilization uterinetransfer) procedure entails the transcervical transfer of several 2-6cell (day 2) or 8-16 (day 3) preimplantation embryos to the fundus ofthe uterus (4-5 preimplantation embryos provides optimal success).

Procedures for in vitro fertilization are also described in U.S. Pat.Nos. 6,610,543 6,585,982, 6,544,166, 6,352,997, 6,281,013, 6,196,965,6,130,086, 6,110,741, 6,040,340, 6,011,015, 6,010,448, 5,961,444,5,882,928, 5,827,174, 5,760,024, 5,744,366, 5,635,366, 5,691,194,5,627,066, 5,563,059, 5,541,081, 5,538,948, 5,532,155, 5,512,476,5,360,389, 5,296,375, 5,160,312, 5,147,315, 5,084,004, 4,902,286,4,865,589, 4,846,785, 4,845,077, 4,832,681, 4,790,814, 4,725,579,4,701,161, 4,654,025, 4,642,094, 4,589,402, 4,339,434, 4,326,505,4,193,392, 4,062,942, and 3,854,470, the contents of which arespecifically incorporated by reference for their description of theseprocedures.

Alternatively, patients may elect to have the oocyte, optionallycomprising exogenous, autologous OSC mitochondria, reimplanted andfertilized in vivo using Intrauterine Insemination (IUI). IUI is a wellknown process that involves preparing and delivering a highlyconcentrated amount of active motile sperm directly through the cervixinto the uterus. There are several techniques available for preparingthe sperm for IUI. First, sperm is separated from seminal fluid. Onemethod of sperm separation is known as “Density Gradient Separation”. Inthis technique, motile sperm are separated from dead sperm and othercells through the use of viscous solution. After preparation, the spermconcentrate is placed through the cervix into the uterus by using athin, flexible catheter and fertilization of the reimplanted oocytefollows.

Culture Medium

Physiologically compatible solutions can be formulated or supplementedwith effective amounts of bioenergetic agents or functional derivativesthereof for conducting the methods of the invention (e.g., IVF,cryopreservation, gamete preparation, cell and/or embryo washing orculture). Cell culture medium, embryo culture medium andcryopreservation solutions, for example, are well known in the art andcan be formulated or supplemented as needed using standard methods knownin the art for the preparation of physiological solutions. Commerciallyavailable medium for the preparation and handling of gametes for invitro fertilization includes G-IVF™ PLUS, available from Invitrolife,which is a bicarbonate buffered medium containing human serum albuminand gentamicin as an antibacterial agent. SAGE Media™ maturation mediumfor oocytes contains sodium chloride, potassium chloride, sodiumbicarbonate, glucose, sodium pyruvate, phenol red, gentamicin,nonessential and essential amino acids, magnesium sulfate, sodiumphosphate, calcium chloride, D-calcium pantothenate, chlorine chloride,folic acid, i-inositol, nicotimamide, pyridoxine, HCL, riboflavin, andthiamine, supplemented with a final concentration of 75 mIU/ml FSH and75 mIU/ml LH supplemented with a final concentration of 75 mIU/ml FSHand 75 mIU/ml LH. Media products for freezing and containment of humanblastocysts include SAGE Media™ equilibration solution and vitrificationsolution available from SAGE In Vitro Fertilization, Inc. Ovarianfollicle maturation medium is also well known in the art and described,for example, by Telfer et al., Hum Reprod 2008 23: 1151-1158, thecontents of which are expressly incorporated herein by reference. TheSAGE Media™ equilibration solution is a MOPS buffered solution ofmodified HTF containing nonessential and essential amino acids,gentamicin sulfate (0.01 g/L), 7.5% (v/v) each of DMSO and ethyleneglycol and 12 mg/mL human albumin. The vitrification solution is a MOPSbuffered solution of modified human tubal fluid (HTF) containingnonessential and essential amino acids, gentamicin sulfate (0.01 g/L),and 15% (v/v) each of DMSO and ethylene. SAGE blastocyst medium isformulated for use with in vitro fertilization procedures involving theculture of human embryos from the compaction phase on day 3 ofdevelopment to the blastocyst stage and consists of sodium chloride,potassium chloride, potassium phosphate, magnesium sulfate, calciumlactate, sodium bicarbonate, glucose, sodium pyruvate, taurine,glutathione, alanyl-glutamine, L-asparagine, L-aspartic acid, glycine,L-proline, L-serine, L-arginine, L-cystine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine,L-tryptophan, L-tyrosine, L-valine, D-calcium pantothenate, cholinechloride, folic acid, I-inositol, nicotinamide, pyridoxine, riboflavin,thiamine, gentamicin and phenol red. SAGE cleavage medium is formulatedfor use with in vitro fertilization procedures involving the culture ofcleavage stage human embryos and consists of sodium chloride, potassiumchloride, magnesium sulfate, calcium lactate, sodium bicarbonate,glucose, sodium pyruvate, alanyl-glutamine, taurine, L-asparagine,L-aspartic acid, glycine, L-proline, L-serine, sodium citrate, EDTA,gentamicin and phenol red. SAGE fertilization medium is formulated foruse with in vitro procedures involving the fertilization of humanoocytes and consists of sodium chloride, potassium chloride, magnesiumsulfate, potassium phosphate, calcium lactate, sodium bicarbonate,glucose, sodium pyruvate, alanyl-glutamine, taurine, L-Asparagine,L-Aspartic acid, glycine, L-Proline, L-Serine, sodium citrate, EDTA,gentamicin and phenol red. Several products are commercially availablefrom LIFEGLOBAL® Group LLC, USA, including solutions for embryo washingand handling (consisting of sodium chloride, potassium chloride, calciumchloride, potassium phosphate, magnesium sulfate, sodium bicarbonate,glucose, lactate na salt, sodium pyruvate, amino acids, edta, gentamicinphenol red, and HEPES, optionally enriched with selected non-essentialamino acids); oocyte retrieval and washing (consisting of sodiumchloride, potassium chloride, calcium chloride, potassium phosphate,magnesium sulfate, sodium bicarbonate, glucose, lactate na salt, sodiumpyruvate, gentamicin, phenol red, and HEPES); embryo culture from day 1to the blastocyst stage (consisting of sodium chloride, potassiumchloride, calcium chloride, potassium phosphate, magnesium sulfate,sodium bicarbonate, glucose, lactate na salt, sodium pyruvate, aminoacids, edta, gentamicin and phenol red); maintenance of embryos duringthe biopsy procedure (consisting of sodium chloride, potassium chloride,potassium phosphate, sodium bicarbonate, glucose sodium lactate, sodiumpyruvate, amino acids, edta, phenol red, gentamicin sulfate, HEPES,sucrose and human serum albumin); embryo freezing (consisting of sodiumchloride, calcium chloride, potassium chloride, potassium phosphate,magnesium chloride, sodium phosphate, and human serum albumin) andembryo thawing (consisting of sodium chloride, calcium chloride,potassium chloride, potassium phosphate, magnesium chloride, sodiumphosphate, human serum albumin and optionally 1,2-propanediol andsucrose). EARLY CLEAVAGE MEDIA™ (ECM®) is available from IrvineScientific, Santa Ana, Calif., USA, and is intended for use in culturinghuman gametes during fertilization (IVF) and growth of embryos throughday 3 of development. This solution consists of glucose sodium, pyruvatesodium, lactate (d/1), sodium chloride, potassium chloride, magnesiumsulfate, calcium chloride, sodium bicarbonate, alanyl-glutamine,taurine, sodium citrate, edta, disodium, dehydrate, phenol red,gentamicin, and sulfate. A culture medium for human gametes and embryosduring fertilization and growth of embryos up to day 5/6 of developmentis also available from Irvine Scientific, Santa Ana, Calif., USA, andconsists of sodium chloride, potassium chloride, potassium phosphate,calcium chloride, magnesium sulfate, sodium bicarbonate, sodiumpyruvate, glucose, sodium lactate, EDTA, dipeptide, alanyl-glutamine,phenol red, gentamicin, alanine, asparagines, aspartic acid, glutamicacid, glycine, proline, serine, arginine, cystine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan, tyrosine and valine.

Accordingly, appropriate selection or formulation of medium for use inconducting assisted reproductive technologies (e.g., IVF) is routinelypracticed by physicians and clinical laboratories. An effective amountof any one or more of the bioenergetic agents or functional derivativesthereof can be added to the medium of interest either prior to, duringor following protocols conducted in connection with assistedreproductive technologies, which are well known in the art. Theconcentration, time and other conditions used will be optimized toachieve maximum exposure of the bioenergetic agents or functionalderivatives thereof to the desired tissues or cells of interest,including, but not limited to, ovarian tissue, oocytes, OSCs orderivatives thereof (e.g., cytoplasm or isolated mitochondria) orpreimplantation embryos. Tissues and/or cells of interest can betreated, ex vivo, using medium comprising bioenergetic agents orfunctional derivatives thereof prior to or during procedures associatedwith assisted reproductive technologies known in the art including, butnot limited to, oocyte or OSC maturation and collection, ovarianfollicle maturation, ovarian tissue or ovarian cell grafting, ovariantissue or ovarian cell transplantation, cryopreservation, in vitrofertilization, as well as the culture of human oocytes, zygotes andpreimplantation embryos.

The present invention also provides methods of producing an oocyte,comprising culturing a stem cell, including but not limited to, an OSC,embryonic stem cell, skin stem cell, pancreatic stem cell, and inducedpluripotent stem cell (iPS cell) in the presence of a bioenergetic agentor a functional derivative thereof, under conditions sufficient todifferentiate the stem cell into an oocyte.

Stem cells can undergo self-renewing cell division to give rise tophenotypically and genotypically identical daughters for an indefinitetime and ultimately can differentiate into at least one final cell type.Stem cells are defined as cells that have extensive, and perhapsindefinite, proliferation potential that differentiate into several celllineages, and that can repopulate tissues upon transplantation. Thequintessential stem cell is the embryonic stem (ES) cell, as it hasunlimited self-renewal and multipotent differentiation potential. Thesecells are derived from the inner cell mass of the blastocyst, or can bederived from the primordial germ cells from a post-implantation embryo(embryonic germ cells or EG cells). ES and EG cells have been derivedfrom mouse, non-human primates and humans. When introduced into mouseblastocysts or blastocysts of other animals, ES cells can contribute toall tissues of the mouse (animal). When transplanted in post-natalanimals, ES and EG cells generate teratomas, which again demonstratestheir multipotency.

Somatic stem cells have been identified in most organ tissues.Accordingly, in some embodiments, the stem cells useful for the oocytedifferentiation and/or maturation culture methods described hereininclude, but are not limited to OSCs, mesenchymal stem cells,bone-marrow derived stem cells, hematopoietic stem cells, chrondrocyteprogenitor cells, skin stem cells (e.g., epidermal stem cells),gastrointestinal stem cells, neural stem cells, hepatic stem cells,adipose-derived mesenchymal stem cells, pancreatic progenitor and/orstem cells, hair follicular stem cells, endothelial progenitor cells andsmooth muscle progenitor cells.

An “induced pluripotent stem (iPS) cell” is a cell that exhibitscharacteristics similar to embryonic stem cells (ESCs) including, forexample, unlimited self renewal in vitro, a normal karyotype, acharacteristic gene expression pattern including stem cell marker geneslike Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) and stemcell-specific antigen 3 and 4 (SSEA3/4), and the capacity todifferentiate into specialized cell types (Hanna et al., Science 2007318: 1920-1923; Meissner A. et al. Nat Biotechnol 2007 25(10): 1177-81,Okita K. et al. Nature 2007 448(7151): 313-7, Takahashi K. et al. Cell2007 131(5): 861-72, Wernig M. et al. Nature 2007 448(7151): 318-24, YuJ. et al. Science 2007 318(5858): 1917-20, and Park, I. H. et al. Nature2008 451(7175): 141-6. The state of the art generation of iPS cells fromfibroblast cultures has been described in Takahashi, Okita, Nakagawa,Yamanaka Nature Protocols (2007) 2(12).

Conditions for the differentiation and/or maturation of cells, includingstem cells, progenitor cells and reprogrammed cells into oocytes areknown in the art and are described, for example, by Danner S. et al. MolHum Reprod. 2007 Jan.; 13(1): 11-20, Dyce P. W. et al. PLoS One.2011;6(5), Dyce P. W. et al. Stem Cells Dev. 2011 May; 20(5): 809-19,Linher K. et al. PLoS One. 2009 Dec. 14;4(12), Dyce P. W. et al. NatCell Biol. 2006 Apr.;8(4): 384-90, Panula S. et al. Hum Mol Genet. 2011Feb. 15;20(4): 752-62, Park T. S. et al., Stem Cells 2009 Apr.;27(4):783-95, Hua J. et al., Stem Cells Dev. 2008 Jun.;17(3):399-411,Aflatoonian B. et al. Reproduction 2006 Nov.;132(5): 699-707, Ko K. etal. Semin Reprod Med. 2006 Nov.;24(5): 322-9, Ko K. et al. Front Biosci.2010 Jan. 1;15: 46-56, Psathaki O. E. et al. Stem Cells Dev. 2011 Mar.8. [Epub ahead of print], and Hubner K. et al. Science 2003 May23;300(5623): 1251-6, the contents of which are expressly incorporatedherein by reference. In particular, methods described by Telfer E. etal. Hum Reprod. 2008;23(5): 1151-8 describing a two-step serum-freeculture system to support development of human oocytes from primordialfollicles in the presence of activin can be used together with thebioenergetic agents or functional derivatives thereof An effectiveamount of any one or more of the bioenergetic agents or functionalderivatives thereof can be added to the culture medium of interesteither prior to, during or following oocyte differentiation and/ormaturation protocols, which are well known in the art. Theconcentration, time and other conditions used will be optimized toachieve maximum exposure of the bioenergetic agents or functionalderivatives thereof to the stem cells and oocyte derivatives thereof.

Methods of Improving Fertility and/or Restoring Reproductive Function

Bioenergetic agents and functional derivatives thereof can be used in avariety of therapeutic applications for the treatment of infertility,reproductive disorders or symptoms of reproductive aging in femalesubjects. In some instances, the menopausal female subjects can be in astage of either peri- or post-menopause, with said menopause caused byeither normal (e.g., aging) or pathological (e.g., surgery, disease,ovarian damage) processes. Restoration of reproductive (e.g., ovarian)function can relieve adverse symptoms and complications associated withmenopause, including, but not limited to, somatic disorders such asosteoporosis, cardiovascular disease, somatic sexual dysfunction, hotflashes, vaginal drying, sleep disorders, depression, irritability, lossof libido, hormone imbalances, and the like, as well as cognitivedisorders, such as loss of memory; emotional disorders, depression, andthe like.

Thus, the present invention provides methods for improving fertility ina female subject comprising administering a bioenergetic agent or afunctional derivative thereof, in an amount effective to improve oocyteand/or OSC de novo production, quality and/or ovulated oocyte yield.

The present invention also provides methods of in vitro fertilizationcomprising the steps of:

a) administering to a female subject a bioenergetic agent or afunctional derivative thereof, in an amount effective to improve oocyteand/or OSC de novo production, quality and/or ovulated oocyte yield;

b) obtaining an oocyte from the female subject (including obtaining theoocyte from an OSC or tissue of a female subject e.g., an in vitroderived or matured oocyte); and

c) fertilizing the oocyte in vitro to form a zygote. Step b) and/or stepc) can further comprise incubating the oocyte or source thereof with abioenergetic agent or a functional derivative thereof. Administration ofthe bioenergetic agent can occur before oocyte, OSC or ovarian tissueharvest and continue throughout the procedure, including aftertransferring the zygote, or a preimplantation stage embryo into theuterus of the female subject (or a surrogate female subject) andcontinuing, or initiating, administration of the bioenergetic agent tothe pregnant female subject.

The present invention also provides methods of restoring ovarianfunction in a female subject in need thereof comprising administering atherapeutically effective amount of a bioenergetic agent or a functionalderivative thereof, thereby restoring ovarian function in the femalesubject. In general, restoring ovarian function provides restoration ofreproductive health benefits, including but not limited to, normalhormone production, menstrual cycling and adequate oocyte and OSCreserves. The female subject in need of restored ovarian function canhave, for example, premature ovarian failure.

The present invention also provides methods for sustaining, maintainingand/or prolonging embryonic development in a pregnant female subject inneed thereof comprising administering a therapeutically effective amountof a bioenergetic agent or a functional derivative thereof.

In some embodiments, methods of the invention have beneficial effects onpregnancy outcomes, which include but are not limited to, a greaternumber of viable embryo transfers, increased fertilization and pregnancyrates (e.g., with corresponding decreases in the number of implantedembryos), decreased rates of multiple births, and improved implantation,gestation and embryogenesis, collectively referred to herein as“pregnancy success” when compared to a reference standard. A standardcan permit one of skill in the art to determine the amount of pregnancysuccess by evaluating the relative increase and/or decrease of one ormore parameters (e.g., viable embryo transfers, fertilization andpregnancy rates, numbers of implanted embryos, multiple births,implantation, and length of gestation and embryogenesis). A standardserves as a reference level for comparison, such that results can benormalized to an appropriate standard in order to infer the presence,absence or extent of a pregnancy success. In one embodiment, a standardis obtained from the same individual as that being tested, at an earliertime point (i.e., before initiation of treatment with a bioenergeticagent or functional derivative thereof). Thus, one or more parameterscontributing to pregnancy success from a patient is compared to previoushistory associated with the same parameters, which acts as a reference.This type of standard is generally the most accurate for diagnostic,prognostic and efficacy monitoring purposes, since a majority of factorswill remain relatively similar in one individual over time. The standardshould ideally be obtained prior to the onset of treatment. However, astandard can be obtained from an individual after the treatment as itcan still provide information about improvement or regression of thetreatment. A standard can also be obtained from another individual or aplurality of individuals, wherein a standard represents an average levelpregnancy success among a population of individuals with or withouttreatment. Thus, the level of pregnancy success in a standard obtainedin this manner is representative of an average level in the givenpopulation, such as a general population of females of reproductive age.

The present invention also provides methods of preparing a tissue orcell thereof from a female subject for harvest (e.g., removal from thebody), comprising administering an effective amount of a bioenergeticagent or a functional derivatives thereof, to the female subject,thereby preparing said tissue or cell thereof from the female subjectfor harvest. The tissue can be, for example, ovary, ovarian follicle,bone marrow and peripheral blood and the cell can be, for example, anoocyte or an OSC. Harvested tissues and/or cells of interest canoptionally be treated, ex vivo, using medium comprising bioenergeticagents or functional derivatives thereof prior to or during proceduresassociated with assisted reproductive technologies known in the artincluding, but not limited to, oocyte or OSC maturation and collection,ovarian follicle maturation, grafting, transplantation,cryopreservation, in vitro fertilization, as well as the culture ofhuman embryos and zygotes.

By “an effective amount” or “therapeutically effective amount” is meantthe amount of a required a bioenergetic agent or a functional derivativethereof, or composition comprising the agent to ameliorate the symptomsof a disorder (e.g., infertility, age related reproductive decline)relative to an untreated patient. The effective amount of agents used topractice the present invention for therapeutic treatment variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount” or “therapeutically effectiveamount.”

Generally, doses of the compounds of the present invention would be fromabout 0.01 mg/kg per day to about 2000 mg/kg per day. In one embodiment,0.01, 0.05, 0.1, 0.5, 1, 3, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 1800,1900, 2000 mg of a bioenergentic agent (e.g., Sirt1 activator, CD38inhibitor) is administered to a subject. Effective doses range fromabout 0.01 mg/kg per day to about 2000 mg/kg per day, where the bottomof the range is any integer between 0.01 and 1999, and the top of therange is any integer between 0.02 and 1000. It is expected that dosesranging from about 5 to about 2000 mg/kg will be suitable—depending onthe specific bioenergetic agent used. Lower doses will result fromcertain forms of administration, such as intravenous administration andpharmaceutical. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels of acomposition of the present invention.

The invention provides methods of administering pharmaceuticalcompositions and formulations comprising bioenergetic agents orfunctional derivatives thereof. In alternative embodiments, thecompositions of the invention are formulated with a pharmaceuticallyacceptable carrier. In alternative embodiments, the pharmaceuticalcompositions and formulations of the invention can be administeredparenterally, topically, orally or by local administration, such as byaerosol or transdermally. The pharmaceutical compositions can beformulated in any way and can be administered in a variety of unitdosage forms depending upon the condition or disease (e.g., type ofreproductive disorder) and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., the latest edition of Remington'sPharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

Bioenergetic agents or functional derivatives thereof can beadministered alone or as a component of a pharmaceutical formulation(composition). The compounds may be formulated for administration, inany convenient way for use in human or veterinary medicine. Wettingagents, emulsifiers and lubricants, such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, release agents, coatingagents, sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Formulations of the compositions of the invention include those suitablefor intradermal, inhalation, oral/ nasal, topical, parenteral, rectal,and/or intravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,bioenergetic agents, or functional derivatives thereof) which can becombined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration, e.g., intradermal or inhalation. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect, e.g. improved oocyte or OSC productionand quality and/or increased yield of ovulated oocytes.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents and preserving agents. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., bioenergeticagents, or functional derivatives thereof) in admixture with excipientssuitable for the manufacture of aqueous suspensions, e.g., for aqueousintradermal injections. Such excipients include a suspending agent, suchas sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

In one embodiment, oil-based pharmaceuticals are used for administrationof nucleic acid sequences of the invention. Oil-based suspensions can beformulated by suspending an active agent in a vegetable oil, such asarachis oil, olive oil, sesame oil or coconut oil, or in a mineral oilsuch as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No.5,716,928 describing using essential oils or essential oil componentsfor increasing bioavailability and reducing inter- and intra-individualvariability of orally administered hydrophobic pharmaceutical compounds(see also U.S. Pat. No. 5,858,401). The oil suspensions can contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol.Sweetening agents can be added to provide a palatable oral preparation,such as glycerol, sorbitol or sucrose. These formulations can bepreserved by the addition of an antioxidant such as ascorbic acid. As anexample of an injectable oil vehicle, see Minto et al., J. Pharmacol.Exp. Ther. 1997 281: 93-102.

Pharmaceutical formulations of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent. In alternative embodiments, these injectable oil-in-wateremulsions of the invention comprise a paraffin oil, a sorbitanmonooleate, an ethoxylated sorbitan monooleate and/or an ethoxylatedsorbitan trioleate.

In practicing this invention, the pharmaceutical compounds can also beadministered by in intranasal, intraocular and intravaginal routesincluding suppositories, insufflation, powders and aerosol formulations(for examples of steroid inhalants, see e.g., Rohatagi J. Clin.Pharmacol. 1995 35: 1187-1193; Tjwa et al., Ann. Allergy Asthma Immunol.1995 75: 107-111). Suppositories formulations can be prepared by mixingthe drug with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at body temperatures and will thereforemelt in the body to release the drug. Such materials are cocoa butterand polyethylene glycols.

In practicing this invention, the pharmaceutical compounds can bedelivered transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, paints, powders, and aerosols.

In practicing this invention, the pharmaceutical compounds can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection of drug whichslowly release subcutaneously; see Rao J. Biomater Sci. Polym. Ed. 19957: 623-645; as biodegradable and injectable gel formulations, see, e.g.,Gao Pharm. Res. 1995 12: 857-863; or, as microspheres for oraladministration, see, e.g., Eyles J. Pharm. Pharmacol. 1997 49: 669-674.

In practicing this invention, the pharmaceutical compounds can beparenterally administered, such as by intravenous (IV) administration oradministration into a body cavity or directly into the ovary. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

The pharmaceutical compounds and formulations of the invention can belyophilized. The invention provides a stable lyophilized formulationcomprising a composition of the invention, which can be made bylyophilizing a solution comprising a pharmaceutical of the invention anda bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose ormixtures thereof.

The compositions and formulations of the invention can be delivered bythe use of liposomes. By using liposomes, particularly where theliposome surface carries ligands specific for target cells, or areotherwise preferentially directed to a specific organ, one can focus thedelivery of the active agent into target cells in vivo. See, e.g., U.S.Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed J. Microencapsul. 1996 13:293-306; Chonn Curr. Opin. Biotechnol. 1995 6: 698-708; Ostro Am. J.Hosp. Pharm. 1989 46: 1576-1587.

The formulations of the invention can be administered for prophylacticand/or therapeutic treatments. In alternative embodiments, fortherapeutic applications, compositions are administered to a subject inneed of improved oocyte or OSC production and quality and/or increasedyield of ovulated oocytes in an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of the reproductivedisorder or its complications, e.g., infertility, menopause, prematureovarian failure; this can be called a therapeutically effective amount.

The amount of pharmaceutical composition adequate to accomplish this isa therapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones J. Steroid Biochem. Mol. Biol. 1996 58: 611-617;Groning Pharmazie 199651: 337-341; Fotherby Contraception 1996 54:59-69; Johnson J. Pharm. Sci. 1995 84: 1144-1146; Rohatagi Pharmazie1995 50: 610-613; Brophy Eur. J. Clin. Pharmacol. 1983 24: 103-108; thelatest Remington's, supra. The state of the art allows the clinician todetermine the dosage regimen for each individual patient, active agentand disease or condition treated. Guidelines provided for similarcompositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, i.e., dose schedule and dosage levels,administered practicing the methods of the invention are correct andappropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, the degree and amount of cholesterolhomeostasis generated after each administration, and the like. Theformulations should provide a sufficient quantity of active agent toeffectively treat, prevent or ameliorate conditions, diseases orsymptoms, e.g., improve oocyte or OSC production and quality and/orincrease yield of ovulated oocytes.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in topical or oral administration or administering by powders,spray or inhalation. Actual methods for preparing parenterally ornon-parenterally administrable formulations will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington's, supra.

The present invention is additionally described by way of the followingillustrative, non-limiting Examples that provide a better understandingof the present invention and of its many advantages.

EXAMPLES

In Examples 1-6, validated protocols are employed to demonstrate thatOSCs can be reliably isolated from tissues of healthy young women andpropagated in vitro for use in subsequent clinical procedures. InExample 7, CR during adulthood is shown to improve oocyte quality andyield in female mice on the verge of reproductive failure due toadvancing maternal age. In Example 8, similar to CR, bioenergeticfactors are shown to increase mitochondrial parameters in OSCs. Thefollowing examples are put forth for illustrative purposes only and arenot intended to limit the scope of what the inventors regard as theirinvention.

Example 1 FACS—Based Protocol for OSC Isolation

The VASA antibody used by Zou et al., Nat Cell Biol 2009 11: 631-636 toisolate mouse OSCs by immunomagnetic sorting is a rabbit polyclonalagainst the last 25 amino acids of the COOH-terminus of human VASA(DDX4) (ab13840; Abcam, Cambridge, Mass., USA). This region shares 96%overall homology with the corresponding region of mouse VASA (MVH). Forcomparative studies, a goat polyclonal antibody against the first 145amino acids of the NH₂-terminus of human VASA (AF2030; R&D Systems) wasused, which shares 91% overall homology with the corresponding region ofmouse VASA.

Immunofluorescence analysis of young adult (2-month-old) mouse ovariesusing either antibody showed an identical pattern of VASA expressionthat was restricted, as expected, to oocytes (FIG. 1a ). Each antibodywas then used for immunomagnetic sorting of dispersed young adult mouseovary tissue (Zou et al., Nat Cell Biol 2009 11: 631-636). For eachpreparation of cells, ovaries from 4 mice were pooled and dissociated bymincing followed by a two-step enzymatic digestion involving a 15-minuteincubation with 800 U/ml collagenase [type IV; prepared in Hank'sbalanced salt solution minus calcium and magnesium (HBSS)] followed by a10-minute incubation with 0.05% trypsin-EDTA. Digestions were carriedout in the presence of 1 μg/ml DNase-I (Sigma Aldrich Corporation, St.Louis, Mo., USA) to minimize stickiness within the cell preparations,and trypsin was neutralized by addition of 10% fetal bovine serum (FBS;HYCLONE®, GE Healthcare Bio-Sciences, Pittsburgh, Pa., USA). Ovariandispersates were filtered through a 70-μm nylon mesh and blocked in asolution composed of 1% fatty-acid free bovine serum albumin (BSA; SigmaAldrich Coroporation, St. Louis, Mo., USA) with either 1% normal goatserum (Millipore; for subsequent reactions using ab13840 againstVASA-COOH) or 1% normal donkey serum (Sigma Aldrich Corporation, St.Louis, Mo., USA; for subsequent reactions using AF2030 against VASA-NH₂)in HBSS for 20 minutes on ice. Cells were then reacted for 20 minutes onice with a 1:10 dilution of VASA antibody that recognizes either theCOOH terminus (ab13840) or NH₂ terminus (AF2030). Afterwards, cells werewashed 2 times in HBSS and incubated for 20 minutes on ice with a 1:10dilution of either goat anti-rabbit IgG-conjugated microbeads (Miltenyi;ab13840 detection) or biotin-conjugated donkey anti-goat IgG (Santa CruzBiotechnology; AF2030 detection) followed by incubation withstreptavidin-conjugated microbeads (Miltenyi). After one additional washin HBSS, the cell preparations were loaded onto MACS columns andseparated according to manufacturer's specifications (Miltenyi). Forexperiments to visualize potential antibody-bead interaction withindividual oocytes, adult female mice were superovulated by injection ofpregnant mare serum gonadotropin (PMSG, 10 IU; Sigma AldrichCorporation, St. Louis, Mo., USA) followed by human chorionicgonadotropin (hCG, 10 IU; Sigma Aldrich Corporation, St. Louis, Mo.,USA) 46-48 hours later. Oocytes were collected from oviducts 15-16 hoursafter hCG injection, denuded of cumulus cells using hyaluronidase(Irvine Scientific, Santa Ana, Calif., USA) and washed with human tubalfluid (HTF; Irvine Scientific, Santa Ana, Calif., USA) supplemented withBSA. Dispersed ovarian cells or isolated oocytes were blocked andincubated with primary antibodies against VASA as described above. Afterwashing in HBSS, cells were reacted with species-appropriate secondaryantibodies conjugated to 2.5-μm Dynabeads (INVITROGEN®). Suspensionswere placed into 1.5 ml Eppendorf tubes for separation using a DynalMPC®-S Magnetic Particle Concentrator.

No cells were obtained in the bead fraction when the VASA-NH₂ antibodywas used; however, 5-8 μm cells bound to the magnetic beads wereobserved when the VASA-COOH antibody was used (FIG. 1b ). Analysis ofthese cells revealed a germline gene expression pattern consistent withthat reported for OSCs isolated previously by Zou et al., Nat Cell Biol2009 11: 631-636 using immunomagnetic sorting (FIG. 2). Althoughisolated oocytes assessed in parallel using the VASA-COOH antibody werealways detected in the non-immunoreactive wash fraction (FIG. 1b ),additional marker analysis of the VASA-positive cell fraction obtainedby immunomagnetic sorting revealed several oocyte-specific mRNAsincluding Nobox, Zp3 and Gdf9 (FIG. 2). These findings indicate thatwhile oocytes do not exhibit cell surface expression of VASA whenanalyzed as individual entities (FIG. 1b ), oocytes are nonetheless acontaminating cell type following immunomagnetic sorting of OSCs fromdispersed ovary tissue. This outcome most likely reflects either anon-specific physical carry-over of oocytes during the beadcentrifugation steps or reactivity of cytoplasmic VASA in plasmamembrane-compromised (damaged) oocytes with the COOH antibody. Eithercase would be alleviated by use of FACS.

The reactivity of each antibody with dispersed mouse ovarian cells wasnext assessed by FACS. For each experiment, ovarian tissue (mouse: 4ovaries pooled; human: 10×10×1 mm thick, cortex only) was dissociated,blocked and reacted with primary antibody (ab13840 for VASA-COOH orAF2030 for VASA-NH₂) as described above. After washing with HBSS, cellswere incubated with a 1:500 dilution of goat anti-rabbit IgG conjugatedto Alexa Fluor 488 (INVITROGEN®; ab13840 detection) or donkey anti-goatIgG conjugated to Alexa Fluor 488 (INVITROGEN®; AF2030 detection) for 20minutes on ice, and washed with HBSS. Labeled cells were then filteredagain (35-μm pore diameter) and sorted by FACS using a FACSARIA II®cytometer (BD Biosciences, San Jose, Calif., USA), gated againstnegative (unstained and no primary antibody) controls. Propidium iodidewas added to the cell suspension just prior to sorting for dead cellexclusion. Freshly-isolated VASA-positive viable cells were collectedfor gene expression profiling, assessment of teratoma formation capacityor in-vitro culture. For some experiments, cells were fixed in 2%neutral-buffered paraformaldehyde (PFA) and permeabilized with 0.1%Triton-X100 prior to reaction with primary antibody against the NH₂terminus of VASA (AF2030) and detection by FACS after reaction withdonkey anti-goat IgG conjugated to Alexa Fluor 488. For re-sortexperiments, viable cells were reacted with VASA-COOH antibody (ab13840)and sorted by FACS after reaction with a goat anti-rabbit IgG conjugatedto allophcocyanin (APC) (Jackson Immunoresearch). Resultant APC-positive(VASA-COOH positive) viable cells were then either left intact or fixedand permeabilized prior to incubation with VASA-NH₂ antibody (AF2030),followed by incubation with donkey anti-goat IgG conjugated to AlexaFluor 488 and FACS analysis.

In agreement with the magnetic bead sorting results, viableVASA-positive cells were obtained only when the COOH antibody was used(FIG. 1c ). However, if the ovarian cells were permeabilized prior toFACS, a VASA-positive cell population was obtained using the NH₂antibody (FIG. 1c ). Furthermore, if the viable VASA-positive cellsisolated by FACS using the COOH antibody were permeabilized andre-sorted, the same cell population was recognized by the VASA-NH₂antibody (FIG. 1d ). As a final means to confirm validity of this OSCisolation method, fractions of cells at each step of the protocol wereassessed by gene expression analysis using a combination of markers forgerm cells (Blimp1/Prdm1, Stella/Dppa3 , Fragilis/Ifitm3, Tert, Vasa,Dazl) and oocytes (Nobox, Zp3, Gdf9). To obtain cells for FACS, ovariantissue was minced and enzymatically digested using collagenase andtrypsin, passed through a 70-μm filter to remove large tissue clumps,and then passed through a 35-μm filter to obtain a final fraction ofcells. Every fraction of cells through each step of the protocol, withthe exception of the VASA-positive viable cell fraction obtained byFACS, expressed all germline and oocyte markers (FIG. 1f ). While theFACS-sorted VASA-positive cell fraction expressed all germline markers,no oocyte markers were detected (FIG. 1f ). Thus, unlike the oocytecontamination observed when OSCs are isolated by immunomagnetic sortingusing the VASA-COOH antibody (see FIG. 2), use of this same antibodywith FACS provides a superior strategy to obtain adult ovary-derived OSCfractions free of oocytes.

Example 2 Isolation of OSCs From Human Ovaries

With written informed consent, ovaries were surgically removed from 6female patients between 22-33 (28.5±4.0) years of age with GenderIdentity Disorder for sex reassignment at Saitama Medical Center. Theouter cortical layer was carefully removed, vitrified and cryopreserved(Kagawa et al., Reprod. Biomed. 2009 Online 18: 568-577; FIG. 12).Briefly, 1 mm-thick cortical fragments were cut into 100-mm² (10×10 mm)pieces, incubated in an equilibration solution containing 7.5% ethyleneglycol (EG) and 7.5% dimethylsulfoxide (DMSO) at 26 C for 25 minutes,and then incubated in a vitrification solution containing 20% EG, 20%DMSO and 0.5 M sucrose at 26 C for 15 minutes prior to submersion intoliquid nitrogen. For experimental analysis, cryopreserved ovarian tissuewas thawed using the Cryotissue Thawing Kit (Kitazato Biopharma) andprocessed immediately for histology, xenografting or OSC isolation.Using the COOH antibody, viable VASA-positive cells between 5-8 p.m indiameter were also consistently isolated by FACS from human ovariancortical tissue biopsies of all patients between 22-33 years of age,with a percent yield (1.7%±0.6% VASA-positive versus total viable cellssorted; mean±SEM, n=6) that was comparable to the yield of OSCs fromyoung adult mouse ovaries processed in parallel (1.5%±0.2% VASA-positiveversus total viable cells sorted; mean±SEM, n=15). This percent yield isthe incidence of these cells in the final pool of viable single cellssorted by FACS, which represents a fraction of the total number of cellspresent in ovaries prior to processing. To estimate the incidence ofOSCs per ovary, the genomic DNA content per ovary of 1.5-2 month-oldmice was determined (1,774.44±426.15 μg; mean±SEM, n=10) and dividedinto genomic DNA content per fraction of viable cells sorted per ovary(16.41±4.01 μg; mean±SEM, n=10). Assuming genomic DNA content per cellis equivalent, how much of the total ovarian cell pool is represented bythe total viable sorted cell fraction obtained after processing wasdetermined. Using this correction factor, the incidence of OSCs perovary was estimated to be 0.014%±0.002% [0.00926× (1.5%±0.2%)]. Withrespect to OSC yield, this number varied across replicates but between250 to slightly over 1,000 viable VASA-positive cells per adult ovarywere consistently obtained after FACS of dispersates initially preparedfrom a pool of 4 ovaries.

Analysis of freshly-isolated VASA-positive cells from both mouse andhuman ovaries (FIG. 3a, 3b ) revealed a similar size and morphology(FIG. 3c, 3d ), and a matched gene expression profile rich in markersfor early germ cells (Saitou et al., Nature 2002 418: 293-300; Ohinataet al., Nature 2005 436: 207-213; Dolci et al., Cell Sci. 2002 115:1643-1649) (Blimp1, Stella, Fragilis and Tert; FIG. 3e ). These resultsagree with the morphology and gene expression profile of mouse OSCsreported in the scientific literature (Zou et al., Nat Cell Biol 200911: 631-636, Pacchiarotti et al., Differentiation 2010 79: 159-170).

To further define characteristic features of VASA-positive cellsobtained from adult ovaries, mouse OSCs were tested using an in-vivoteratoma formation assay. This was important since a recent study hasreported the isolation of Oct3/4-positive stem cells from adult mouseovaries that possess the teratoma-forming capacity of embryonic stemcells (ESCs) and induced pluripotent stem cells (iPSCs) (Gong et al.,Fertil. Steril. 2010 93: 2594-2601). Ovaries were collected from a totalof 100 young adult female mice, dissociated and subject to FACS forisolation of VASA-COOH positive viable cells, as described above.Freshly isolated mouse OSCs were injected subcutaneously near the rearhaunch of NOD/SCID female mice (1×10⁵ cells injected per mouse). As acontrol, mouse embryonic stem cells (mESC v6.5) were injected intoage-matched female mice in parallel (1×10⁵ cells injected per recipientmouse). Mice were monitored weekly for up to 6 months for tumorformation.

As expected, 100% of the mice transplanted with mouse ESCs used as apositive control developed teratomas within 3 weeks; however, noteratomas were observed in mice transplanted in parallel withVASA-positive cells isolated from adult mouse ovaries, even at 24 weekspost-transplant (FIGS. 3f-k ). Thus, while OSCs express numerous stemcell and primitive germ cell markers (Zou et al., Nat Cell Biol 2009 11:631-636, Pacchiarotti et al., Differentiation 2010 79: 159-170; see alsoFIG. 1f and FIG. 3e ), these cells are clearly distinct from other typesof pluripotent stem cells described to date.

Example 3 Generation of Oocytes from FACS-purified mouse OSCs

The ability of FACS-purified mouse OSCs, engineered to express GFPthrough retroviral transduction (after their establishment asactively-dividing germ cell-only cultures in vitro) to generate oocytesfollowing transplantation into ovaries of adult female mice wasassessed. To ensure the outcomes obtained were reflective of stableintegration of the transplanted cells into the ovaries and also were notcomplicated by pre-transplantation induced damage to the gonads, 1×10⁴GFP-expressing mouse OSCs were injected into ovaries of non-chemotherapyconditioned wild-type recipients at 2 months of age and animals weremaintained for 5-6 months prior to analysis. Between 7-8 months of age,transplanted animals were induced to ovulate with exogenousgonadotropins (a single intraperitoneal injection of PMSG (10 IU)followed by hCG (10 IU) 46-48 hours later), after which their ovariesand any oocytes released into the oviducts were collected. Ovulatedcumulus-oocyte complexes were transferred into HTF supplemented with0.4% BSA, and assessed by direct fluorescence microscopy for GFPexpression. Developing follicles containing GFP-positive oocytes werereadily detectable, along with follicles containing GFP-negativeoocytes, in ovaries of females that received GFP-expressing mouse OSCsinitially purified by FACS (FIG. 4a ).

After oviductal flushing, complexes containing expanded cumulus cellssurrounding centrally-located oocytes both lacking and expressing GFPwere observed. Mixing of these complexes with sperm from wild-type malesresulted in fertilization and development of preimplantation embryos.For in-vitro fertilization (IVF), the cauda epididymides and vasdeferens were removed from adult wild-type C57BL/6 male mice and placedinto HTF medium supplemented with BSA. Sperm were obtained by gentlysqueezing the tissue with tweezers, capacitated for 1 hour at 37° C.,and then mixed with cumulus-oocyte complexes (1-2×10⁶ sperm/ml in HTFmedium supplemented with BSA) for 4-5 hours. Inseminated oocytes werethen washed of sperm and transferred to fresh medium. At 4-5 hourspost-insemination, oocytes (fertilized and unfertilized) weretransferred to 50 μl drops of KSOM-AA medium (Irvine Scientific, Santa,Ana, Calif., USA), and the drops were covered with mineral oil tosupport further preimplantation embryonic development. Light andfluorescence microscopic examination was performed every 24 hours for atotal of 144 hours to monitor embryo development to the hatchingblastocyst stage (Selesniemi et al., Proc. Natl. Acad. Sci. USA 2011108: 12319-12324). Ovarian tissue harvested at the time of ovulatedoocyte collection from the oviducts was fixed and processed forimmunohistochemical detection of GFP expression using a mouse monoclonalantibody against GFP (sc9996; Santa Cruz Biotechnology) along with theMOM™ kit (Vector Laboratories), as detailed previously (Lee et al. J.Clin. Oncol. 2007 25: 3198-3204). Ovaries from non-transplantedwild-type female mice and from TgOG2 transgenic female mice served asnegative and positive controls, respectively, for GFP detection.

Preimplantation embryos derived from fertilized GFP-positive eggsretained GFP expression through the hatching blastocyst stage (FIG. 4b-d). From the 5 adult wild-type female mice transplanted withGFP-expressing OSCs 5-6 months earlier, a total of 31 cumulus-oocytecomplexes were retrieved from the oviducts, 23 of which successfullyfertilized to produce embryos. The presence of cumulus cells around eachoocyte made it impossible to accurately determine the numbers ofGFP-negative versus GFP-positive oocytes ovulated. However, evaluationof the 23 embryos produced following in-vitro fertilization (IVF)revealed that 8 were GFP-positive, with all 5 mice tested releasing atleast one egg at ovulation that fertilized to produce a GFP-positiveembryo. These findings indicate that OSCs purified by VASA-COOHantibody-based FACS, like their previously reported counterpartsisolated by immunomagnetic sorting (Zou et al., Nat Cell Biol 2009 11:631-636), generate functional oocytes in vivo. However, our data alsoshow that chemotherapy conditioning prior to transplantation is not, aspreviously reported (Zou et al., Nat Cell Biol 2009 11: 631-636),required for OSCs to engraft and generate functional oocytes in adultovary tissue.

Example 4 In-vitro Characterization of Candidate Human OSCs

Using parameters described previously for in-vitro propagation of mouseOSCs (Zou et al., Nat Cell Biol 2009 11: 631-636), adult mouse and humanovary-derived VASA-positive cells were placed into defined cultures withmitotically-inactive mouse embryonic fibroblasts (MEFs) as feeders.Briefly, cells were cultured in MEMα (INVITROGEN®) supplemented with 10%FBS (HYCLONE®, GE Healthcare Bio-Sciences, Pittsburgh, Pa., USA), 1 mMsodium pyruvate, 1 mM non-essential amino acids, 1×-concentratedpenicillin-streptomycin-glutamine (INVITROGEN®), 0.1 mMβ-mercaptoethanol (Sigma), 1×-concentrated N-2 supplement (R&D Systems),leukemia inhibitory factor (LIF; 10³ units/ml; Millipore), 10 ng/mlrecombinant human epidermal growth factor (rhEGF; INVITROGEN®), 1 ng/mlbasic fibroblast growth factor (bFGF; INVITROGEN®), and 40 ng/ml glialcell-derived neurotropic factor (GDNF; R&D Systems). Cultures wererefreshed by the addition of 40-80 μl of new medium every other day, andcells were re-plated on fresh MEFS every two weeks. To assessproliferation, MEF-free OSC cultures were treated with 10 μM BrdU (SigmaAldrich Corporation, St. Louis, Mo., USA) for 48 hours prior to fixationin 2% PFA for dual immunofluorescence-based detection of BrdUincorporation (mitotically-active cells) and VASA expression (germcells), as described (Zou et al., Nat Cell Biol 2009 11: 631-636). Nosignal was detected if primary antibodies were omitted or replaced withan equivalent dilution of normal rabbit serum (not shown).

Freshly-isolated OSCs could be established as clonal lines, and thecolony formation efficiency for human OSCs not seeded onto MEFs rangedfrom 0.18% to 0.40%. Accurate assessment of colony formation efficiencycould not be performed using MEFs as initial feeders, the latter ofwhich greatly facilitates establishment of mouse and human OSCs invitro. After 10-12 weeks (mouse) or 4-8 weeks (human) in culture,actively-dividing germ cell colonies became readily apparent (FIG. 5).Once established and proliferating, the cells could be re-established asgerm cell-only cultures in the absence of MEFs without loss ofproliferative potential. Dual analysis of VASA expression andbromodeoxyuridine (BrdU) incorporation in MEF-free cultures revealedlarge numbers of double-positive cells (FIG. 6a-d ), confirming thatadult mouse and human ovary-derived VASA-positive cells were activelydividing. At this stage, mouse cells required passage at confluenceevery 4-5 days with cultures split 1:6-1:8 (estimated doubling time of14 hours; FIG. 6e ). The rate of mouse OSC proliferation wasapproximately 2-3 fold higher than that of human germ cells maintainedin parallel, the latter of which required passage at confluence every 7days with cultures split 1:3-1:4. Cell surface expression of VASAremained detectable on the surface of more than 95% of the cells aftermonths of propagation (FIG. 6f ). The remaining cells not detected byFACS using the VASA-COOH antibody were large (35-50 μm in diameter)spherical cells spontaneously produced by mouse and human OSCs duringculture, which exhibited cytoplasmic expression of VASA and aredescribed in detail in Example 5.

Gene expression analysis of the cultured cells confirmed maintenance ofearly germline markers (FIG. 6g ). Several oocyte-specific markers werealso detected in these cultures. Levels of mRNA were assessed by RT-PCRusing a SuperScript® VILO™ cDNA Synthesis Kit (INVITROGEN®) and PlatinumTaq polymerase (INVITROGEN®). All products were sequenced to confirmidentity. Sequences of forward and reverse primers used, along withGenBank accession numbers of the corresponding genes, are provided inTable 3 (mouse) Table 4 (human).

TABLE 3 PCR primers used to analyze gene expression in mouse cell and tissue samples. Primer sequences Accession (5′to 3′; F, forward; Size Gene number R, reverse) (bp) Blimp1 NM_007548F: CGGAAAGCAACCCAAAGCAATAC 483 (SEQ ID NO: 2) R: CCTCGGAACCATAGGAAACATTC(SEQ ID NO: 3) Stella NM_139218 F: CCCAATGAAGGACCCTGAAAC 354(SEQ ID NO: 4) R: AATGGCTCACTGTCCCGTTCA (SEQ ID NO: 5) FragilisNM_025378 F: GTTATCACCATTGTTAGTGTCATC 151 (SEQ ID NO: 6)R: AATGAGTGTTACACCTGCGTG (SEQ ID NO: 7) Tert NM_009354F: TGCCAATATGATCAGGCACTCG 305 (SEQ ID NO: 8) R: ACTGCGTATAGCACCTGTCACC(SEQ ID NO: 9) Vasa NM_ F: GGAAACCAGCAGCAAGTGAT 213 001145885(SEQ ID NO: 10) R: TGGAGTCCTCATCCTCTGG (SEQ ID NO: 11) Dazl NM_010021F: GTGTGTCGAAGGGCTATGGAT 328 (SEQ ID NO: 12) R: ACAGGCAGCTGATATCCAGTG(SEQ ID NO: 13) Msy2 NM_016875 F: CCTCCCCACTTTCCCATAAT 235(SEQ ID NO: 14) R: AATGGGTGGGGAAGAAAAAC (SEQ ID NO: 15) Sycp3 NM_011517F: AGCAGAGAGCTTGGTCGGG 100 (SEQ ID NO: 16) R: TCCGGTGAGCTGTCGCTGTC(SEQ ID NO: 17) Dmc1 NM_ F: CTCACGCTTCCACAACAAGA  81 010059.2(SEQ ID NO: 18) R: TCTCGGGGCTGTCATAAATC (SEQ ID NO: 19) Nobox NM_130869F: CCCTTCAGTCACAGTTTCCGT 379 (SEQ ID NO: 20) R: GTCTCTACTCTAGTGCCTTCG(SEQ ID NO: 21) Lhx8 NM_010713 F: CGTCAGTCCCAACCATTCTT 157(SEQ ID NO: 22) R: TTGTTGGTGAGCATCCATGT (SEQ ID NO: 23) Gdf9 NM_008110F: TGCCTCCTTCCCTCATCTTG 709 (SEQ ID NO: 24) R: CACTTCCCCCGCTCACACAG(SEQ ID NO: 25) Zp1 NM_009580 F: GTCCGACTCCTGCAGAGAAC 208(SEQ ID NO: 26) R: TGATGGTGAAGCGCTGATAG (SEQ ID NO: 27) Zp2 NM_011775F: AAGGTCTTGAGCAGGAACGA 152 (SEQ ID NO: 28) R: GGGTGGAAAGTAGTGCGGTA(SEQ ID NO: 29) Zp3 NM_011776 F: CCGAGCTGTGCAATTCCCAGA 183(SEQ ID NO: 30) R: AACCCTCTGAGCCAAGGGTGA (SEQ ID NO: 31) β-actinNM_007393 F: GATGACGATATCGCTGCGCTG 440 (SEQ ID NO: 32)R: GTACGACCAGAGGCATACAGG (SEQ ID NO: 33)

TABLE 4 PCR primers used to analyze gene expression inhuman cell and tissue samples. Acces- Primer sequences sion (5′to 3′; F, Size Gene number  forward; R, reverse) (bp) Blimp1 NM_F: AAACATGACCGGCTACAAGACCCT 332 001198 (SEQ ID NO: 34)R: GGCACACCTTGCATTGGTATGGTT (SEQ ID NO: 35) Stella NM_F: AGCAGTCCTCAGGGAAATCGAAGA 276 199286 (SEQ ID NO: 36)R: TATGGCTGAAGTGGCTTGGTGTCT (SEQ ID NO: 37) Fragilis NM_F: ATGTCGTCTGGTCCCTGTTC 205 021034 (SEQ ID NO: 38)R: GGGATGACGATGAGCAGAAT (SEQ ID NO: 39) Tert NM_F: AGACGGTGTGCACCAACATCTACA 271 198253 (SEQ ID NO: 40)R: TGTCGAGTCAGCTTGAGCAGGAAT (SEQ ID NO: 41) Vasa NM_F: TTGTTGCTGTTGGACAAGTGGGTG 283 024415 (SEQ ID NO: 42)R: GCAACAAGAACTGGGCACTTTCCA (SEQ ID NO: 43) Dazl NM_F: TCGAACTGGTGTGTCCAAAGGCTA 260 001190811 (SEQ ID NO: 44)R: TAGGATTCATCGTGGTTGTGGGCT (SEQ ID NO: 45) Msy2 NM_F: ACCCTACCCAGTACCCTGCT 248 015982 (SEQ ID NO: 46)R: GCAAGAAAAGCAACCAGGAG (SEQ ID NO: 47) Sycp3 NM_F: TATGGTGTCCTCCGGAAAAA 238 001177949 (SEQ ID NO: 48)R: AACTCCAACTCCTTCCAGCA (SEQ ID NO: 49) Nobox NM_F: ATAAACGCCGAGAGATTGCCCAGA 375 001080413 (SEQ ID NO: 50)R: AAGTCTGGTCAGAAGTCAGCAGCA (SEQ ID NO: 51) Lhx8 NM_F: CAAGCACAATTTGCTCAGGA 230 001001933 (SEQ ID NO: 52)R: GGCACGTAGGCAGAATAAGC (SEQ ID NO: 53) Gdf9 NM_F: TCACCTCTACAACACTGTTCGGCT 344 005260 (SEQ ID NO: 54)R: AAGGTTGAAGGAGGCTGGTCACAT (SEQ ID NO: 55) Zp1 NM_F: CGCCATGTTCTCTGTCTCAA 219 207341 (SEQ ID NO: 56)R: CGTTTGTTCACATCCCAGTG (SEQ ID NO: 57) Zp2 NM_ F: TCTTCTTCGCCCTTGTGACT217 003460 (SEQ ID NO: 58) R: CTCAGGGTGAGCTTTTCTGG (SEQ ID NO: 59) Zp3NM_ F: AGCAGGACCCAGATGAACTCAACA 274 001110354 (SEQ ID NO: 60)R: AAGCCCACTGCTCTACTTCATGGT (SEQ ID NO: 61) β- NM_F: CATGTACGTTGCTATCCAGGC 250 actin 001101 (SEQ ID NO: 62)R: CTCCTTAATGTCACGCACGAT (SEQ ID NO: 63)

To extend the mRNA analyses of Blimp1, Stella and Fragilis,immunofluorescence analysis of these three classic primitive germlinemarkers was performed (Saitou et al., Nature 2002 418: 293-300; Ohinataet al., Nature 2005 436: 207-213). For analysis of cultured OSCs, cellswere washed with 1×-concentrated phosphate-buffered saline (PBS), fixedin 2% PFA for 45 minutes at 20° C., washed 3 times with PBS-T (PBScontaining 0.01% Triton-X100) and incubated for 1 hour at 20° C. inblocking buffer (PBS containing 2% normal goat serum and 2% BSA). Thecells were then incubated for 1 hour at 20° C. with a 1:100 dilution ofone of the following primary antibodies: a biotinylated mouse monoclonalagainst BLIMP1 (ab81961, Abcam, Cambridge, Ma., USA), a rabbitpolyclonal against STELLA (ab19878; Abcam, Cambridge, Ma.) or a rabbitpolyclonal against FRAGILIS (mouse: ab15592, human: ab74699; Abcam,Cambridge, Ma., USA).

Cells were washed and incubated for 30 minutes at 20 C with a 1:500dilution of streptavidin-conjugated Alexa Fluor 488 (INVITROGEN®; BLIMP1detection) or goat anti-rabbit IgG conjugated to Alexa Fluor 488 (STELLAand FRAGILIS detection) in the presence of rhodamine-phalloidin(INVITROGEN®). Cells were washed, incubated with4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma AldrichCorporation, St. Louis, Mo., USA) and washed 3 additional times beforeimaging. No signal was detected if primary antibody was omitted orreplaced with normal serum (not shown).

For assessment of oocytes generated in vitro by mouse and human OSCs,individual oocytes were collected from culture supernatants, washed,fixed with 2% PFA containing 0.5% BSA for 45 minutes at 37° C., washedand blocked for 1 hour at 20° C. in PBS containing 0.5% BSA and either5% normal goat serum (VASA or LHX8 detection) or 1% normal donkey serum(c-KIT detection). After blocking, oocytes were incubated for 2 hours at20° C. with a 1:100 dilution (in PBS with 0.5% BSA) of one of thefollowing primary antibodies: a goat polyclonal against c-KIT (sc1494,Santa Cruz Biotechnology), a rabbit polyclonal against VASA (ab13840,Abcam, Cambridge, Ma., USA) or a rabbit polyclonal against LHX8(ab41519, Abcam, Cambridge, Ma., USA). Cells were then washed andincubated with a 1:250 dilution of goat anti-rabbit IgG conjugated toAlexa Fluor 568 (INVITROGEN®; VASA detection) or Alexa Fluor 488 (LHX8detection), or a 1:250 dilution of donkey anti-goat IgG conjugated toAlexa Fluor 488 (c-KIT detection). Cells were washed, incubated withDAPI and washed 3 additional times before imaging. No signal wasdetected if primary antibody was omitted or replaced with normal serum.

For these latter experiments, detection of oocyte-specific expression ofVASA, c-KIT and, for human ovaries, LHX8 in ovarian tissue sectionsserved as a positive control. Mouse and human ovarian tissue was fixedin 4% PFA, paraffin-embedded and sectioned (6-μm) prior to hightemperature antigen retrieval using 0.01 M sodium citrate buffer (pH6.0). After cooling, sections were washed and blocked for 1 hour at 20°C. using TNK buffer (0.1 M Tris-HCl, 0.55 M NaCl, 0.1 mM KCL, 0.5% BSA,and 0.1% Triton-X100 in phosphate-buffered saline) containing either 1%normal goat serum (VASA-COOH or LHX8 detection) or 1% normal donkeyserum (VASA-NH₂ or c-KIT detection). Sections were then incubated with a1:100 dilution of primary antibody (in TNK buffer with 1% normal serum)overnight at 4 C, washed in PBS, and incubated for 30 minutes at 20° C.with a 1:500 dilution of goat anti-rabbit IgG conjugated to Alexa Fluor568 (VASA-COOH detection in human ovary), goat anti-rabbit IgGconjugated to Alexa Fluor 488 (detection of VASA-COOH in mouse ovary orLHX8) or donkey anti-goat IgG conjugated to Alexa Fluor 488 (c-KIT orVASA-NH₂ detection). After washing with PBS, sections were cover-slippedusing Vectashield containing DAPI (Vector Labs). No signal was detectedif primary antibody was omitted or replaced with normal serum.

All three proteins were easily and uniformly detected in mouse (FIG. 6h) and human (FIG. 6i ) OSCs maintained in vitro. Notably, detection ofFRAGILIS in these cells agrees with a recent study reporting that thisprotein can also be used to isolate OSCs from mouse ovaries byimmunomagnetic bead sorting (Zou et al., Stem Cells Dev. 2011 doi:10.1089/scd.2011.0091).

Example 5 In-vitro Oogenic Capacity of Candidate Human OSCs

Consistent with results from others (Pacchiarotti et al.,Differentiation 2010 79: 159-170), mouse OSCs cultured in vitrospontaneously generated large (35-50 μm in diameter) spherical cellsthat by morphology (FIG. 7a ) and gene expression analysis (FIG. 7b, c )resembled oocytes. Peak levels of in-vitro oogenesis from mouse OSCswere observed within 24-48 hours after each passage (FIG. 7d ), followedby a progressive decline to nearly non-detectable levels each time OSCsregained confluence. Parallel analysis of VASA-positive cells isolatedfrom adult human ovaries and maintained in vitro revealed that thesecells, like mouse OSCs, also spontaneously generated oocytes as deducedfrom both morphological (FIG. 7f ) and gene expression (FIG. 7c, g )analyses. The kinetics of in-vitro oogenesis from human OSCs differedslightly from mouse OSCs in that peak levels of oocyte formation wereobserved at 72 hours after each passage (FIG. 7e ). In addition todetection of many widely accepted oocyte markers (Vasa, c-Kit, Nobox,Lhx8, Gdf9, Zp1, Zp2, Zp3; (Suzumori et al., Mech. Dev. 2002 111:137-141; Rajkovic et al., Science 2004 305: 1157-1159; Pangas et al.,Proc. Natl. Acad. Sci. USA 2006 103: 8090-8095; Elvin et al., Mol.Endocrinol. 1999 13: 1035-1048; Zheng et al., Semin. Reprod. Med. 200725: 243-251), mouse and human OSC-derived oocytes also expressed thediplotene oocyte stage-specific marker Msy2 (FIG. 7c ). MSY2 is amammalian homologue of Xenopus FRGY2, a germ cell-specific nucleicacid-binding Y-box protein that is essential for meiotic progression andgametogenesis in both sexes (Gu et al., Biol. Reprod. 1998 59:1266-1274; Yang et al., Proc. Natl. Acad. Sci. USA 2005 102: 5755-5760).Through empirical testing of commercially-available antibodies usingadult human ovarian cortical tissue as a positive control, four suchantibodies against oocyte markers were identified that specificallyreacted with immature oocytes present in adult human ovaries (VASA,c-KIT, MSY2, LHX8; FIG. 8); all four of these proteins were alsodetected in oocytes generated by human OSCs in vitro (FIG. 7g ).

The presence of mRNA encoding the meiotic marker MSY2 in oocytes newlyformed from human OSCs in vitro prompted us to next explore theprospects of meiotic entry in these cultures. Immunofluorescenceanalysis of attached (non-oocyte germline) cells 72 hours after passageidentified cells with punctate nuclear localization of themeiosis-specific DNA recombinase, DMC1, and the meiotic recombinationprotein, synaptonemal complex protein 3 (SYCP3) (FIG. 7h ). Bothproteins are specific to germ cells and are necessary for meioticrecombination (Page et al., Annu. Rev. Cell Dev. Biol. 2004 20: 525-558;Yuan et al., Science 2002 296: 1115-1118; Kagawa et al., FEBS 1 2010277: 590-598).

Chromosomal DNA content analysis of human OSC cultures 72 hours afterpassage was determined. Cultured mouse (48 hours after passage) or human(72 hours after passage) OSCs were collected by trypsinization, washedand resuspended in ice-cold PBS, and counted with a hemocytometer. Afterfixation in ice-cold 70% ethanol for 1 hour, cells were washed inice-cold PBS and incubated with 0.2 mg/ml RNase-A for 1 hour at 37° C.Propidium iodide was then added (10 μg/ml final), and ploidy status wasdetermined using a FACSARIA II® cytometer (BD Biosciences, San Jose,Calif., USA). As a control somatic cell line, these experiments wererepeated using human fetal kidney fibroblasts (KEK 293, INVITROGEN®).This analysis revealed the presence of an expected diploid (2n) cellpopulation; however, peaks corresponding to 4n and 1n populations ofcells were detected, the latter being indicative of germ cells that hadreached haploid status (West et al., Stem Cells Dev. 2011 20: 1079-1088)(FIG. 7i ). In actively-dividing cultures of fetal human kidneyfibroblasts analyzed as controls in parallel, only 2n and 4n populationsof cells (FIG. 9a ) were detected. Comparable outcomes were observedfollowing FACS-based chromosomal analysis of mouse OSC cultures (FIG. 9b).

Example 6 Human OSCs Generate Oocytes in Human Ovarian Cortical TissueIn Vivo

To confirm and extend the in-vitro observations of putative oogenesisfrom candidate human OSCs, in two final experiments VASA-positive cellsisolated from adult human ovaries were stably transduced with a GFPexpression vector (GFP-hOSCs) to facilitate cell tracking. For celltracking experiments, human OSCs were transduced using a retrovirus toobtain cells with stable expression of GFP (GFP-hOSCs). Briefly, 1 μg ofpBabe-Gfp vector DNA (Addgene plasmid repository #10668) was transfectedas per the manufacturer's protocol (Lipofectamine, INVITROGEN®) into thePlatinum-A retroviral packaging cell line (Cell Biolabs). Viralsupernatant was collected 48 hours after transfection. Transduction ofhuman OSCs was performed using fresh viral supernatant facilitated bythe presence of polybrene (5 μg/ml; Sigma Aldrich Corporation, St.Louis, Mo., USA). After 48 hours, the virus was removed and replacedwith fresh OSC culture medium. Human OSCs with expression of GFP werepurified by FACS following an initial 1 week of expansion, and thepurified cells were expanded for additional 2 weeks before a secondround of FACS purification to obtain GFP-hOSCs for human ovarian tissuere-aggregation or xenografting experiments.

In the first experiment, approximately 1×10⁵ GFP-hOSCs were thenre-aggregated with dispersed adult human ovarian cortical tissue. Humanovarian cortex was dissociated and washed as described above, andincubated with 35 μg/mlphytohemaglutannin (PHA; Sigma) plus 1×10⁵GFP-hOSCs for 10 minutes at 37° C. The cell mix was pelleted bycentrifugation (9,300× g for 1 minute at 20° C.) to create the tissueaggregate, which was placed onto a Millicell 0.4 um culture plate insert(Millipore) contained in a 6-well culture dish with 1 ml of OSC culturemedium. Aggregates were incubated at 37° C. in 5% CO₂-95% air, andlive-cell GFP imaging was performed 24, 48 and 72 hours later.

Numerous GFP-positive cells were observed, as expected, throughout there-aggregated tissue (FIG. 10a ). The aggregates were then placed inculture and assessed 24-72 hours later by direct (live cell) GFPfluorescence. Within 24 hours, several very large (≥50-μm) single cellswere also observed in the aggregates, many of which were enclosed bysmaller GFP-negative cells in tightly compact structures resemblingfollicles; these structures remained detectable through 72 hours (FIG.10b, c ). These findings indicated that GFP-expressing human OSCsspontaneously generated oocytes that became enclosed by somatic(pregranulosa/granulosa) cells present in the adult human ovariandispersates.

Next, GFP-hOSCs were injected into adult human ovarian cortical tissuebiopsies, which were then xenografted into NOD/SCID female mice (n=40grafts total). Ovarian cortical tissue pieces (2×2×1 mm) wereindividually injected with approximately 1.3×10³ GFP-hOSCs using a 10-μlNanoFil syringe with a 35-gauge beveled needle (World PrecisionInstruments). Recipient NOD/SCID female mice were anesthetized and asmall incision was made along the dorsal flank for subcutaneousinsertion of the human ovarian tissue, essentially as described(Weissman et al., Biol. Reprod. 1999 60: 1462-1467; Matikainen et al.,Nature Genet. 2001 28: 355-360). Xenografts were removed after 7 or 14days post transplantation, fixed in 4% PFA, paraffin-embedded andserially sectioned (6-μm) for immunohistochemical analysis using a mousemonoclonal antibody against GFP (sc9996; Santa Cruz Biotechnology) (Leeet al., J. Clin. Oncol. 2007 25: 3198-3204). Briefly, high temperatureantigen retrieval was first performed using 0.01 M sodium citrate buffer(pH 6.0). After cooling, sections were incubated for 10 minutes with 3%hydrogen peroxide in methanol to block endogenous peroxidase activity,washed and incubated in streptavidin-biotin pre-block solution as perthe manufacturer's protocol (Vector Laboratories). Sections were thenblocked for 1 hour at 20° C. using TNK buffer containing 1% normal goatserum and incubated overnight at 4 C with a 1:100 dilution of GFPantibody prepared in TNK buffer containing 1% normal goat serum.Sections were then washed, incubated with a 1:500 dilution of goatanti-mouse biotinylated secondary antibody for 30 minutes at 20° C.,washed and reacted with Vectastain ABC reagents (Lab Vision) for 30minutes at 20° C. prior to detection of GFP-positive cells usingdiaminobenzidine (DAKO). Sections were lightly counterstained withhaematoxylin to visualize cell and tissue architecture. Negativecontrols (complete immunohistochemical staining protocol on xenograftedtissues that received vehicle injections) were always run in paralleland did not show a positive signal. To confirm and extend theseobservations, dual immunofluorescence-based detection of GFP and eitherMSY2 (diplotene stage oocyte-specific marker) or LHX8 (early stageoocyte transcription factor) in xenografted human ovarian tissues wasperformed with DAPI counterstaining, as detailed previously in thedescription of immunoanalysis.

Grafts were collected 7 or 14 days later for assessment of GFPexpression. All human ovary grafts contained easily discernibleprimordial and primary follicles with centrally-located GFP-negativeoocytes. Interdispersed among and often adjacent to these follicles,which were presumably present in the tissue prior to GFP-hOSC injection,were other immature follicles containing GFP-positive oocytes (FIG. 10d,f ). Serial section histomorphometric analysis of 3 randomly selectedhuman ovarian tissue biopsies injected with GFP-hOSCs revealed thepresence of 15-21 GFP-positive oocytes per graft 7 days afterxenografting into mice (FIG. 11). As controls, GFP-positive oocytes werenever detected in human ovarian cortical tissue prior to GFP-hOSCinjection (FIG. 10e ) or in xenografts that received mock injections(vehicle without GFP-hOSCs) prior to transplantation into NOD/SCID mice(FIG. 10g ). Dual immunofluorescence-based detection of GFP along witheither the diplotene stage oocyte-specific marker MSY2 (Gu et al., Biol.Reprod. 1998 59: 1266-1274; Yang et al., Proc. Natl. Acad. Sci. USA 2005102: 5755-5760) or the oocyte-specific transcription factor LHX8 (Pangaset al., Proc. Natl. Acad. Sci. USA 2006 103: 8090-8095) identified manydual-positive cells distributed throughout xenografts injected withGFP-hOSCs (FIG. 10h ). As expected, no GFP-positive oocytes weredetected in ovarian tissue prior to GFP-hOSC injection or in xenograftsthat did not receive GFP-hOSC injections (not shown; see FIG. 10e , g);however, these oocytes were consistently positive for LHX8 and MSY2(FIG. 10h ; FIG. 8).

Example 7 Use of OSCs in Autologous Germline Mitochondrial EnergyTransfer “AUGMENT”

FIG. 13 depicts an overview of the use of OSCs as an autologous sourceof female germ cells for derivation of oogenic cytoplasm ormitochondrial fractions that can then be transferred into an oocyte oregg obtained from the same subject prior to or during in vitrofertilization (IVF). The resultant boost in mitochondrial DNA copynumber and ATP-generating capacity in the egg after AUGMENT ensures thatthe egg has ample reserves of ATP for energy-driven events required forsuccessful fertilization and embryonic development. The additionalmitochondria provided to the egg by AUGMENT are derived from the naturalprecursor cell used by the body to produce egg cells. Furthermore, theadditional mitochondria will not produce adverse effects in the egg,based on data showing that healthy embryogenesis proceeds even when theminimal threshold number of mitochondria needed for embryo developmentis exceeded by nearly four-fold (see Wai et al., Biology of Reproduction2010 83: 52-62, FIG. 6). The beneficial effects of heterologousooplasmic transfer reported earlier by Cohen et al., Mol Hum Reprod 19984: 269-80, a procedure which is restricted for human use because itresults in germline genetic manipulation and mitochondrial heteroplasmyin embryos/offspring, indicate that eggs are benefitted by additionalmitochondria.

An exemplary clinical protocol for AUGMENT is as follows. Prior to thestart of standard IVF, the subject will undergo a laparoscopy duringmenstrual cycle days 1-7 to collect up to three pieces (approximately3×3×1 mm each) of ovarian epithelium (ovarian cortical biopsy) from oneovary. During this procedure, 2-3 incisions will be made within theabdomen and a device will be inserted to remove the tissue from an ovaryusing sterile procedures. The tissue collected will be placed in sterilesolution and transported on ice to the GTP compliant laboratory where itwill be cryopreserved until the time of AUGMENT/ICSI. The tissue willremain frozen until the time of enzymatic dissociation. This will serveas the source of autologous OSCs from which mitochondria will bepurified.

Next, OSCs will be isolated and mitochondria will be harvested from theOSCs. After thawing the ovarian cortical biopsied tissue, the tissuewill be minced and placed in solution, containing recombinantcollagenase and recombinant DNasel and homogenized to a single cellsuspension. The suspension will be passed through a cell strainer toprepare a solution of single cells. The single cell suspension will beincubated with an anti-VASA antibody. Labeled cells will then beisolated by fluorescence-activated cell sorting (FACS). Standard slowcooling cryopreservation procedures for freezing aliquots of OSCs willbe used.

Subjects will undergo a standard IVF protocol including baselineevaluation, GnRH antagonist down-regulation and gonadotropinstimulation. Oocyte retrieval will take place within 34-38 hours afterhCG administration and oocytes will be assessed for quality andmaturation state. Mature oocytes will be inseminated by ICSI.

On the day of egg retrieval, the frozen OSC vial for that subject willbe thawed using standard methods. OSCs will be processed to yield amitochondrial pellet (Frezza et al. Nature Protocols 2007 2: 287-295 orPerez et al., Cell Death and Differentiation 2007 3: 524-33. Epub 2006Oct. 13) or as described below in Example 10, where a FACS-based methodis employed to isolate the total mitochondrial population in a tissueand optionally, further isolate the actively respiring mitochondrialpopulation or quantitate the ratio of active to total mitochondria in atissue. Evaluation and activity of the mitochondrial preparation will beassessed and recorded. The mitochondrial pellet will be re-suspended inmedia to a standardized concentration of mitochondrial activity whichimproves oocyte quality. This media containing the mitochondria will beaspirated into a microinjection needle that contains the spermatozoan tobe delivered. Both the mitochondria and spermatozoan will be deliveredtogether into the oocyte by ICSI.

Following fertilization and embryo culture, a maximum of three, grade 1or grade 2 (SART grading system (50)) embryos may be transferred underultrasound guidance after 3 or 5 days of culturing based on theassessment of embryo development. If a pregnancy is confirmed via betahCG testing, then the subject will have subsequent observations atapproximately 6 and 20-weeks gestational age.

Example 8 CR-Induced Mitochondrial Stimulation Improves Oocyte Qualityand Yield in Females with Increasing Age

Restricted caloric intake without malnutrition extends lifespan andattenuates severity of aging-related health complications in manyspecies (Masoro et al., Mech Ageing Dev 2005 126: 913-922; Mair et al.,Annu Rev Biochem 2008 77: 727-754; Fontana et al., Science 2010 328:321-326). A common feature of the CR response appears to be analteration of metabolic regulators that affect mitochondrial dynamicsand accumulated oxidative stress in organs with age (Sohal et al. MechAgeing Dev 1994 74: 121-133, Barja et al. 2002 Ageing Res Rev 1:397-411, Barja et al. Biol Rev Camb Philos Soc 2004 79: 235-251). Forexample, the growth hormone/insulin/insulin-like growth factor-1 axis,mammalian target of rapamycin, AMP-activated protein kinase and sirtuinshave all been implicated as mediators of CR (Fontana et al., Science2010 328: 321-326, Sinclair et al. Mech Ageing Dev 2005 126: 987-1002,Rodgers et al. FEBSLett 2008 582: 46-53, Finley et al. Ageing Res Rev2009 8: 173-188). Several of these pathways reportedly converge onperoxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), atranscriptional regulator that is highly responsive to nutritional cues.Among its actions, PGC-1α promotes adaptation to energy deficiency bymodulating expression of genes involved in mitochondrial respiration(Fontana et al., Science 2010 328: 321-326, Sinclair et al. Mech AgeingDev 2005 126: 987-1002, Rodgers et al. 2008 FEBSLett 582: 46-53, Finleyet al. Ageing Res Rev 2009 8: 173-188, Rodgers J T, et al. Nature 2005434: 113-118, Lin et al. Cell Metab 2005 1: 361-370). Surprisingly,deletion of PGC-1α in mice produces only subtle phenotypes, althoughseveral metabolic abnormalities manifest much more robustly upon achallenge such as acute fasting (Lin et al. Cell 2004 119: 121-135,Arany Z, et al. Cell Metab 2005 1: 259-271, Leone T C, et al. PLoS Biol2005 3: 672-687). However, no studies have tested the functionalrelationship between PGC-1α and CR in any tissue with age by subjectingPgc-1α-null mice to a reduced calorie diet. Accordingly, a 4-yearinvestigation was conducted to elucidate whether CR during adulthoodwithout or with manipulation of PGC-1α influences oocyte quality infemale mice on the verge of reproductive failure due to advancingmaternal age.

Yield, maturational status and post-fertilization developmentalcompetency of oocytes obtained from 12-month-old (aged) female micereturned to an ad-libitum (AL) diet for 1 month following 7.5 months ofdietary CR (CR-AL-fed) initiated in a stepwise fashion at 3.5 months ofage were first evaluated. This protocol was based on prior work showingthat female mice maintained on CR during adulthood continue to breed anddeliver offspring into advanced ages after their return to an AL diet(Selesniemi et al. 2008 Aging Cell 7: 622-629). Mice were superovulatedby injection of pregnant mare serum gonadotropin (PMSG, 10 IU; SigmaAldrich Corporation, St. Louis, Mo., USA) followed by human chorionicgonadotropin (hCG, 10 IU; Sigma Aldrich Corporation, St. Louis, Mo.,USA) 46-48 hours later. Oocytes were collected from oviducts 15-16 hoursafter hCG injection, denuded of cumulus cells using hyaluronidase(Irvine Scientific, Santa Ana, Calif., USA), washed with human tubalfluid (HTF; Irvine Scientific, Santa Ana, Calif., USA) supplemented withBSA (fraction V, fatty acid-free; Sigma Aldrich Corporation, St. Louis,Mo., USA), and classified as MII (first polar body in perivitellinespace), maturation arrested (germinal vesicle breakdown with no polarbody extrusion, or germinal vesicle intact), or degenerated.

In control females allowed to AL feed during the entire study period,the total number of oocytes and number of fully mature oocytes (oocytesthat reached meiotic metaphase II; designated MII) ovulated per femaledecreased significantly between 3 and 12 months of age (FIG. 16A).However, the age-related decline in both total and mature oocyte yieldwas abrogated in 12-month-old female mice maintained on CR (FIG. 16A).

Next, in-vitro fertilization (IVF) and preimplantation embryonicdevelopment rates were assessed. Sperm were collected from the caudaepididymides of male mice into HTF supplemented with BSA and thencapacitated. Denuded MII oocytes or intact cumulus-oocyte complexes weremixed with 1-2×106 sperm/ml in HTF supplemented with BSA for 6-9 hours,washed and transferred to fresh medium. The number of 2-cell embryos wasused to measure IVF success rate, and blastocyst development rates fromthese embryos were recorded. Following analysis of 284 (3-month-oldAL-fed), 93 (12-month-old AL-fed) and 198 (12-month-old CR-AL-fed)oocytes, no differences were observed with respect to in-vitrofertilization (IVF) or preimplantation embryonic development rates (FIG.17). However, because CR improved the yield of MII oocytes per femaleafter an induced ovulation cycle at 12 months of age (FIG. 16A), thenumber of blastocysts obtained following IVF of oocytes obtained fromeach aged CR-AL-fed mouse was similar to that obtained using young miceand significantly higher than that using aged AL-fed mice (FIG. 16B).

To determine if the beneficial effect of CR on maintaining oocyte yieldfrom aging females was related to differences in body weight,superovulation rates in young AL-fed, aged AL-fed and aged CR-AL fedfemales on a mouse-by-mouse basis was assessed. It was observed thatdifferences in oocyte yield per mouse, which were greatest in the agedAL-fed group, were unrelated to variations in body weight among thethree groups of mice (FIG. 19). Also notable was that the reserve ofoocyte-containing follicles in ovaries of both 12-month-old AL-fed andCR-AL-fed females was severely diminished compared to that of3-month-old mice (FIG. 16C). Thus, the ability of CR to maintain a highyield of MII oocytes from aged females does not appear linked to changesin body weight or maintenance of a follicle reserve equivalent in sizeto that of young females.

Next, the quality of MII oocytes collected from aged AL-fed andCR-AL-fed females was studied. Fully mature (MII) oocytes were selectedfor analysis because aging-related defects in oocytes are clearlyevident at this maturational stage and because MII oocytes represent thefertilization-competent egg pool. To this end, chromosomal dynamics,spindle integrity and mitochondrial dynamics were assessed, which arethe important events involved in ensuring developmental competency ofthe egg. A total of 795 mature (MII) oocytes collected from 3-month-oldAL-fed (n=20 mice), 12-month-old AL-fed (n=34 mice) and 12-month-oldCR-AL-fed (n=20 mice) females were fixed individually for chromosomalanalysis using Tarkowski's method (Tarkowski et al. Cytogenetics 1966 5:394-400, Muhlhauser et al. Biol Reprod 2009 80: 1066-1071). Preparationswere stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI;Sigma Aldrich Corporation, St. Louis, Mo., USA) and scored foraneuploidy rates under a fluorescence microscope. In MII oocytescollected from continuously AL-fed females, the incidence of hyperploidy(>20 chromosomes per cell; FIG. 10A) increased significantly fromnon-detectable levels at 3 months of age to nearly 5% at 12 months ofage. In contrast, no hyperploidy was detected in MII oocytes from12-mo-old mice maintained on CR (FIG. 10B). The incidence of hypoploidy(<20 chromosomes per cell) was also significantly elevated in MIIoocytes from 12-month-old versus 3-month-old AL-fed females, and thiswas completely prevented by CR (FIG. 10B). A similar pattern in theincidence of premature sister chromatid separation (PSCS) was observedin mature oocytes among the 3 groups of mice, although these changeswere not statistically significant (FIG. 10B).

Confocal analysis of a-tubulin and DNA distribution were examined.Superovulated oocytes were denuded of cumulus cells, briefly incubatedin Acidified Tyrode's Solution (Irvine Scientific, Santa Ana, Calif.,USA) to soften the zona pellucida, and immunostained using mouseanti-α-tubulin antibody (Sigma Aldrich Corporation, St. Louis, Mo., USA)followed by goat anti-mouse IgG conjugated with Alexa Fluor-488 (LifeTechnologies, Carlsbad, Calif.). Oocytes were mounted using Vectashieldcontaining propidium iodide (PI; Vector Laboratories) and analyzed byconfocal microscopy. Confocal analysis of a-tubulin and DNA distributionrevealed that meiotic spindles in greater than 90% of MII oocytescollected from either 3-month-old AL-fed or 12-month-old CR-AL-fedfemales were regular in shape and size with distinct microtubulemorphology; however, less than 39% of MII oocytes retrieved from12-month-old AL-fed mice exhibited normal meiotic spindles (FIG. 20A andC). Furthermore, while 64% of MII oocytes from 12-month-old AL-fed miceexhibited incomplete or aberrant alignment of chromosomes on themetaphase plate, 25% or less of the MII oocytes collected from either3-month-old AL-fed or 12-month-old CR-AL-fed females exhibitedchromosomal misalignment (FIG. 20B and C).

Whether mitochondrial aggregation, which has been linked to the declinein oocyte quality with advancing age (Tarin et al. Biol Reprod 2001 65:141-150), was affected by caloric intake was assessed. Oocytes weredenuded of cumulus cells, incubated in MitoTracker Red CMRox (LifeTechnologies), and processed for microscopic analysis. Levels of ATP inindividual MII oocytes were determined using a commercially availablebioluminescent assay kit under the manufacturer's specifications (SigmaAldrich Corporation, St. Louis, Mo., USA). Confocal microscopic analysesof MII oocytes stained with MitoTracker revealed that over 90% of MIIoocytes collected from 3-month-old AL-fed females exhibited even anddiffuse cytoplasmic distribution of mitochondria (FIG. 21A and B). Bycomparison, nearly 50% of MII oocytes obtained from 12-month-old AL-fedfemales exhibited extensive mitochondrial aggregation. However, morethan 90% of mature oocytes collected from 12-month-old CR-AL-fed femalesexhibited even and diffuse mitochondrial distribution, resembling thatobserved in MII oocytes retrieved from young females (FIG. 21A and B).Paralleling these changes in mitochondria, the aging-related decline inATP content in oocytes of aged AL-fed females was similarly prevented byadult-onset CR (FIG. 21C).

Finally, gene mutant mice were used to explore if deletion of PGC-1α,which has been linked to the actions of CR in other cell types (Finleyet al. 2009 Ageing Res Rev 8: 173-188, Corton et al. J Gerontol A BiolSci Med Sci 2005 60A: 1494-1509, Anderson et al. Biochim Biophys Acta2009 1790: 1059-1066, López-Lluch et al. Proc Natl Acad Sci 2006 103:1768-1773) and is expressed in oocytes (FIG. 21A and FIG. 23),influences the ability of CR to maintain oocyte quality with age. TotalRNA from five MII oocytes or one ovary was isolated using the RNeasyPlus Micro Kit (Qiagen, Valencia, Calif.) or Tri-Reagent (Sigma AldrichCorporation, St. Louis, Mo., USA), respectively, and reverse transcribed(Superscript II; Life Technologies) with random primers (Promega,Madison, Wis.). The cDNA was amplified by PCR with gene-specificprimers:

TABLE 5 Sequence information for primers used to detect Pgc-1α, Pgc-1βand β-actin mRNA in oocytes andovaries (GenBank Accession numbers are provided) NM_008904 Pgc-1α 5′TCCTCTGACCCCAGACTCAC 3′ Forward (SEQ ID NO: 64) Pgc-1α 5′TAGAGTCTTGGAGCTCCT 3′ Reverse (SEQ ID NO: 65) NM_133249 Pgc-1β 5′AACCCAACCAGTCTCACAGG 3′ Forward (SEQ ID NO: 66) Pgc-1β 5′ATGCTGTCCTTGTGGGTAGG 3′ Reverse (SEQ ID NO: 67) NM_007393 β-Actin  5′GATGACGATATCGCTGCGCTG 3′ Forward (SEQ ID NO: 68) β-Actin  5′GTACGACCAGAGGCATACAGG 3′ Reverse (SEQ ID NO: 69)

Consistent with past studies (Lin et al. Cell 2004 119: 121-135), anabsence of PGC-1α increased mortality in mutant offspring (90 pups of696 total generated by breeding heterozygotes were genotyped asknockouts at day 21). Assessment of null females that survived to 12months (36 of 47 total) showed that PGC-1α deficiency in AL-fed micerecapitulated the beneficial effects of CR on ovulated oocyte yield(FIG. 22B), meiotic spindle formation (FIG. 22C), chromosomal alignment(FIG. 22D) and mitochondrial distribution within the cytoplasm (FIG.22E). At 12 months, AL-fed females lacking PGC-1α exhibited a slightlylarger follicle reserve than their wild-type counterparts, but folliclenumbers remained severely diminished compared to young adult animals ofeither genotype (FIG. 24). No further changes in oocyte numbers perovary (FIG. 24), or in oocyte yield or quality (FIG. 22B-D), wereobserved when mice lacking PGC-1α were subjected to CR.

PGC-1 protein was localized in paraformaldehyde-fixed paraffin-embeddedtissue sections using a rabbit anti-PGC-1 antibody (Calbiochem), asdescribed (Matikainen et al. Nat Genet 2001 28: 355-360). Proteinsamples (10 μg) were assessed by immunoblotting using antibodies againstPGC-1 (Calbiochem) and pan-actin (Neomarkers, Fremont, Calif.) as aloading control. Since levels of PGC-1 protein remained essentiallyunchanged in ovaries of AL- or CR-AL-fed mice with age (FIG. 25), itdoes not appear that CR directly alters PGC-1 gene expression in thisorgan. However, the finding that CR and PGC-1α independently producedthe same outcomes in ovulated oocytes suggests that signaling pathwaysactivated in the two models converge at a common downstream point thatis important to ensuring egg quality.

In summary, this study has uncovered striking beneficial effects ofadult-onset CR on chromosomal, spindle and mitochondrial dynamics inmature oocytes of female mice at ages normally associated with poorreproductive parameters. The present study not only establishes that CRsustains female fertile potential with age through significantimprovements in oocyte chromosomal dynamics, but also identifies PGC-1αas a regulator of oocyte quality. Thus, prevention of oocyte aneuploidyand spindle defects through administration of bioenergetic agents (e.g.,including CR mimetics) provides a means to improve fertility andpregnancy outcomes in women of advanced reproductive age.

Additional Information Regarding Experimental Procedures for CRAnalysis:

An adult-onset CR protocol developed by the National Institute on Agingin their Biomarkers of Aging Study (Turturro et al. J Gerontol A BiolSci Med Sci 1999 54A: B492-B501) was used, in which CR is initiated at3.5 months of age in a stepwise manner over a 2-week period to achieve40% restriction at 4 months of age. Each female was housed individuallyin a conventional (non-ventilated) cage and fed once daily with arationed amount of fortified rodent diet (National Institute on Aging).The fortified rodent diet is supplemented with vitamins and mineralssuch that daily intake of these micronutrients is comparable to that ofcontrol animals with ad libitum (AL) access to the non-fortified(standard) rodent diet. Diet composition is otherwise identical. The CRprotocol was continued until 11 months of age, at which time the micemaintained previously on CR were allowed AL access to standard rodentdiet for 1 month. To confirm that the CR protocol was working asexpected, the weight of each mouse was taken just prior to the start ofthe CR protocol (3 months of age), at the conclusion of the CR protocol(11 months of age) and one month following the return of CR mice to ALfeeding (12 months of age) (FIG. 26). In addition, past studies whichemployed alternating days of fasting and feeding to achieve CR in femalemice reported that aging-related disruption of estrous cyclicity wasdelayed by food restriction (Nelson et al. Biol Reprod 1985 32:515-522). These data, along with more recent observations thatadult-onset CR delays the timing of reproductive failure in female miceas tested in natural mating trials (Selesniemi et al. Aging Cell 2008 7:622-629), support that the approach maintains cyclic production ofreproductive hormones required for normal 45 day estrous cycles. Tofurther confirm this under the feeding protocol employed here to achieveCR, daily vaginal cytological smears were assessed, as described(Felicio et al. Biol Reprod 1986 34: 849-858), to compare estrouscyclicity in aged AL-fed and CR-AL-fed mice over a 30-day period (FIG.27). It is well-established in mice that female reproductive aging isassociated with a shift from typical 45 day estrous cycles to prolongedcycles lasting more than 5 days (Gosden et al. Biol Reprod 1983 28:255-260). For example, the proportion of young adult C57BL/6 miceexhibiting cycles lasting 45 days versus more than 5 days isapproximately 80% to 20%, respectively; however, by 12 months of agenearly two-thirds of female mice exhibit prolonged estrous cyclesindicative of pending ovarian failure (Felicio et al. Biol Reprod 198634: 849-858, Gosden et al. Biol Reprod 1983 28: 255-260; see also FIG.27). All experiments were independently replicated at least 3 times.Quantitative data from experimental replicates were combined and arepresented as the mean±SEM. Statistical comparisons between mean valueswere performed using ANOVA and Student's t-test. P values less than 0.05were considered significant.

Example 9 Bioenergetic Factors Increase Mitochondrial Parameters in OSCs

Female infertility due to chemotherapy, aging and premature ovarianfailure (POF) is due in part to a decline in mitochondrial function inoocytes and OSCs. Accordingly, OSCs from mice were used to screen forsmall compounds that enhance mitochondrial function (“bioenergeticstatus”) in female germline cells. Assays for enhancing mitochondrialfunction included mtDNA content, ATP, NAD+/NADH, mitochondrial mass,mitochondrial membrane potential, and gene expression of knownmitochondrial mass regulators and electron transport chain components.

All the compounds were dissolved in DMSO. For screening purposes thecells were treated with the vehicle (0.001% DMSO) or with 25 or 50 μM ofeach compound (except for berberine, which the concentrations used forthe screen were 5 μM and 25 μM) for 24 hours. For validation of the top6 hits, cells were treated with vehicle (0.001% DMSO) or either 5 or 25μM of each compound for 24 hours. For the gene expression profile, thecells were treated with the concentration that was considered to workbetter in the previous validation assays.

Mitochondrial membrane potential was measured with a fluorescent probe,Tetramethylrhodamine methyl ester (TMRM) (Sigma) as described before(Rolo A. P. Biochim. Biophys. Acta 2003 1637: 127-132). Briefly thecells were loaded with 6.6 μM TMRM in HBSS buffer at 37° C. for 15minutes in the dark. The supernatant was then aspirated, and the cellsreturned to the original volume with KHH. TMRM is a membrane-permeablecationic fluorophore that accumulates electrophoretically inmitochondria in proportion to their mitochondrial membrane potential(Ehrenberg B. V. et al. Biophys. J. 1 1988 53: 785-794). Cellsuspensions (200 μl containing 105 cells) were loaded into 96-wellplates and fluorescence measured using excitation and emissionwavelengths of 485 and 590 nm, respectively. Mitochondrial membranepotential was estimated, taking into account the complete depolarizationcaused by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP).

For mtDNA analysis, total DNA was extracted with DNeasy blood and tissuekit (QIAGEN). mtDNA was amplified using primers specific for themitochondrial cytochrome c oxidase subunit 2 (COX2) gene and normalizedto genomic DNA by amplification of the ribosomal protein s18 (rps18)nuclear gene. Primers were designed using the IDT software (IDT).

ATP content was measured using a luciferase-based assay with acommercial kit according to the manufacturer's instructions (RocheApplied Science, Penzberg, Germany) and normalized to protein content ineach sample.

Mitochondrial mass was evaluated using the fluorescent probe N-nonylacridine orange (NAO), briefly the cells were incubated in culture mediacontaining 10 nM of NAO for 30 minutes at 37° C. in the dark. The cellswere then trypsinized and resuspended in culture media without NAO. TheNAO fluorescence intensity was then determined by flow cytometry on theFACSCALIBUR® (BD Biosciences, San Jose, Calif., USA) using the 488nmlaser.

For gene expression analysis, RNA from skeletal muscle tissue and C2C12cells were extracted with RNeasy mini kit (QIAGEN) according to theinstructions and quantified using the NanoDrop 1000 spectrophotometer(Thermo Scientific). cDNA was synthesized with the iSCRIP cDNA synthesiskit (BioRad) using 200 ng of RNA. Quantitative RT-PCR reactions wereperformed using 1 μM of primers and LIGHTCYCLER® 480 SYBR® Green Master(Roche Applied Science, Penzberg, Germany) on an LIGHTCYCLER® 480detection system (Roche Applied Science, Penzberg, Germany).Calculations were performed by a comparative method (2-ΔΔCT) using actinas an internal control. Primers were designed using the IDT software(IDT).

Several bioenergenic agents known to inhibit CD38 produced activityabove the baseline (DMSO alone) and were scored as a positive result,including apigenin, luteoline, tyrphostin-8, berberine and SRT-1720.Accordingly, these bioenergenic agents were shown to increasemitochondrial parameters in a beneficial way.

Bioenergenic agents, including apigenin, luteolin, berberine, andtyrphostin-8, were shown to raise NAD⁺ levels and increase mitochondrialparameters in a beneficial way. In one embodiment, such agents areuseful in enhancing female germ cell bioenergetics for the treatment offemale infertility associated with chemotherapy, aging and prematureovarian failure.

Example 10 FACS—Based Isolation of Mitochondria

As described in this Example, FACS-based methods can be employed toisolate the total mitochondrial population in a tissue. In addition,FACS-based methods for mitochondrial isolation can employ dual-labelingusing two different fluorescent dyes (mitochondrial membrane potential(MMP)-dependent and MMP-independent) to isolate only the functional(e.g., actively respiring) mitochondrial population or quantitate theratio of functional to total mitochondria in a tissue, cell, lysed cellor fraction derived thereof.

The non-oxidation dependent MitoTracker Green FM (INVITROGEN® M7514)mitochondrial tracking probe, which indicates mitochondrial mass, wasprepared and utilized as described below. MitoTracker stock solution(1-5mg/ml dissolved in anhydrous dimethylsulfoxide (DMSO)) was dilutedin serum free growth medium to reach a working concentration of between25-500 nM. Freshly isolated or thawed OSCs were pelleted bycentrifugation at 300× g for 5 minutes. The supernatant was aspiratedand the cell pellet was resuspended in 200 μl of the diluted MitoTrackerstock solution.

Cells were incubated at 37° C. for 45 minutes, washed in pre-warmed (37°C.) serum free growth medium and pelleted by centrifugation at 300× gfor 5 minutes (alternatively, cells can be lysed prior to incubationwith a probe of interest). Supernatant was aspirated and cells wereresuspended in 100 μl mitochondrial lysis buffer and transferred to aFACS sort tube for lysis by mechanical permeabilization using rapidosmotic shock. Following lysis, cells were equilibrated cells on ice for15-30 minutes, incubated in 200 μl (minimum volume) ice cold PBS andvortexed. As shown in FIG. 36, three distinct populations were observed:residual M7514 positive cells (Cells MT+), high fluorescent mitochondria(functional, Mito MT high), and low expressing mitochondria(non-functional, Mito MT Low). The ratio of functional to non-functionalmitochondria post lysis was approximately 1:1 (1552 mitochondria 743were gated as functional and 716 were gated as non-functional;accumulation the gates are drawn around populations in FIG. 36).Therefore, functional mitochondria can be sorted and collected, withresidual unlysed cells and non-functional mitochondria excluded based onsize and fluorescence intensity. Dual-labeling using multiple probes ora JC-1 probe (red spectrum; INVITROGEN® T3168) can help to furtherdistinguish functional from non-functional mitochondria. Probes for usein dual labeling include, but are not limited, to reduced oxidativestate mitotracker probes (e.g., MitoTracker Red CM-H2XRos (INVITROGEN®M7513), MitoTracker Orange CM-H2TMRos (INVITROGEN® M7511) andaccumulation dependent probes: JC-1 (red spectrum; INVITROGEN® T3168),MitoTracker Deep Red FM (INVITROGEN® M22426) and JC-1 (green spectrum;INVITROGEN® T3168).

Example 11 Mitochondrial Isolation Using Differential Centrifugation

As described in this Example, differential centrifugation procedures canbe employed to isolate and/or fractionate mitochondria present in atissue. The key steps when isolating mitochondria from any tissue orcell are: (i) rupturing of cells by mechanical and/or chemical means,(ii) differential centrifugation at low speed to remove debris andextremely large cellular organelles (SPIN 1), and (iii) centrifugationat a higher speed to isolate and collect mitochondria (SPIN 2).

The tissue is weighed and washed twice with 1.5 ml of a commerciallyavailable Wash Buffer (MitoSciences). The tissue is minced and placed ina pre-chilled Dounce homogenizer. Up to 2.0 ml of a commerciallyavailable Isolation Buffer (MitoSciences) is added. The cells areruptured using the Dounce homogenizer (20-40 strokes), and thehomogenate is transferred to Eppendorf tubes. Each tube is filled to 2.0ml with Isolation Buffer. The homogenate is centrifuged at 1,000 g for10 minutes at 4° C. The supernatant is reserved and transferred into newtubes, each of which is filled to 2.0 ml with Isolation Buffer. Thetubes are centrifuged at 12,000 g for 15 minutes at 4° C. The pellet isreserved. If desired, the supernatant is analysed for quality. Thepellet is washed twice by resuspending in 1.0 ml of Isolation Buffersupplemented with 10 μl of a commercially available protease inhibitorcocktail (MitoSciences). The tubes are centrifuged at 12,000 g for 15minutes at 4° C. After washing, the pellets are combined and resuspendedin 500 μl of Isolation Buffer supplemented with protease inhibitorcocktail. If desired, aliquots are stored at −80° C. until use.

In one approach, mitochondria integrity is tested by Western blotscreening for cytochrome c, porin, or cyclophilin D in the isolatedmitochondria versus in the supernatant fraction using commerciallyavailable antibodies, such as MitoSciences' antibodies MSA06, MSA03, andMSA04. In another approach, mitochondrial samples are probed by Westernblot to detect components of the mitochondrial complex, for example,using the commercially available OXPHOS Complexes Detection cocktail(MitoSciences).

Example 12 Mitochondrial Isolation Using Sucrose Gradient Separation

The protocal employs the following reagents, which are commerciallyavailable: n-dodecyl-β-D-maltopyranoside (Lauryl maltoside; MitoSciencesMS910), Phosphate buffered saline (PBS), Sucrose solutions 15, 20, 25,27.5, 30 and 35%, double distilled water, a protease inhibitor cocktail(MitoSciences), and 13×51 mm polyallomer centrifuge tubes (Beckman326819).

The sucrose gradient separation procedure is a protein subfractionationmethod optimized for mitochondria. This method resolves a sample into atleast 10 fractions. It is possible to separate solubilized whole cellsinto fractions of much lower complexity but when analyzing alreadyisolated mitochondria the fractions are even more simplified. Thesucrose gradient separation technique is designed for an initial samplevolume of up to 0.5 ml at 5 mg/ml protein. Therefore 2.5 mg or less oftotal protein should be used. For larger amounts, multiple gradients canbe prepared or larger scale gradients are made.

The sample is solubilized in a non-ionic detergent. It has beendetermined that at this protein concentration mitochondria arecompletely solubilized by 20 mM n-dodecyl-β-D-maltopyranoside (1% w/vlauryl maltoside). The key to this solubilization process is that themembranes are disrupted while the previously Membrane embeddedmultisubunit OXPHOS complexes remain intact, a step necessary for thedensity based sucrose separation procedure described herein. Oneimportant exception is the pyruvate dehydrogenase enzyme (PDH). In orderto isolate PDH at a protein concentration of 5 mg/ml mitochondria, therequired detergent concentration is only 10 mM (0.5%) lauryl maltoside.The PDH enzyme should also be centrifuged at lower speeds, a centrifugalforce of 16 000 g is maximum for the PDH complex.

To a mitochondrial membrane suspension at 5 mg/ml protein in PBS, laurylmaltoside is added to a final concentration of 1%. This is mixed welland incubated on ice for 30 minutes. The mixture is then centrifuged at72,000 g for 30 minutes. A Beckman Optima benchtop ultracentrifuge isrecommended for small sample volumes. However, at a minimum a benchtopmicrofuge, on maximum speed (e.g., about 16 000 g) should suffice. Aftercentrifugation, the supernatant is collected and the pellet discarded. Aprotease inhibitor cocktail is added to the sample, which is maintainedon ice until centrifugation is performed. In samples very rich inmitochondria the cytochromes in complexes III and IV may give thesupernatant a brown color, which is useful when checking theeffectiveness of the following separation.

A discontinuous sucrose density gradient is prepared by layeringsuccessive decreasing sucrose densities solutions upon one another. Thepreparation and centrifugation of a discontinuous gradient containingsucrose solutions from 15-35% is described in detail below. Thisgradient gives good separation of the mitochondrial OXPHOS complexes(masses ranging from 200 kDa to 1000 kDa). However this setup can bemodified for the separation of a particular complex or for theseparation of larger amounts of material.

The gradient is prepared by layering progressively less dense sucrosesolutions upon one another; therefore the first solution applied is the35% sucrose solution. A steady application of the solutions yields themost reproducible gradient. To aid in this application, a Beckmanpolyallomer tube is held upright in a tube stand. Next a 200 μl pipettetip is placed on the end of a 1000 μl pipette tip. Both snugly fittingtips are held steady by a clamp stand and the end of the yellow tip isallowed to make contact with the inside wall of the tube. Now sucrosesolutions are placed inside the blue tip and fed into the tube slowlyand steadily, starting with the 35% solution (0.25 ml).

Once the 35% solution has drained into the tube, the 30% solution (0.5ml) is be loaded into the tube on top of the 35% solution. Thisprocedure is continued with the 27.5% (0.75 ml), 25% (1.0 ml), 20% (1.0ml) and 15% (1.0 ml), respectively. Enough space is left at the top ofthe tube to add the 0.5 ml sample of solubilized mitochondria.

Once the sucrose gradient is poured discrete layers of sucrose arevisible. Having applied the sample to the top of the gradient the tubeis loaded into the rotor very carefully, and centrifugation begins. Allcentrifugation procedures require a balanced rotor therefore anothertube containing precisely the same mass is generated. In practice thismeans 2 gradients must be prepared although the second gradient need notcontain an experimental sample but could contain 0.5 ml water in placeof the 0.5 ml protein sample.

The polyallomer tubes should be centrifuged in a swinging bucket SW 50.1type rotor (Beckman) at 37,500 rpm (RCF av 132,000× g) for 16 hours 30minutes at 4° C. with an acceleration profile of 7 and decelerationprofile of 7. Immediately after the run the tube should be removed fromthe rotor, taking great care not to disturb the layers of sucrose. Whenseparating a sample rich in mitochondria, discrete colored proteinlayers may be observed. Most often these are Complex III (500 kDa—browncolor) approximately 10 mm from the bottom of the tube and Complex IV(200 kDa—green color) 25 mm from the bottom of the tube. In somecircumstances additional bands can be observed. These are the otherOXPHOS complexes.

For fraction collection, the tube is held steady and upright using aclamp stand. A tiny hole is introduced into the very bottom of the tubeusing a fine needle. The hole is just big enough to allow the sucrosesolution to drip out at approximately 1 drop per second. Fractions ofequal volume are collected in eppendorf tubes below the pierced hole. Atotal of 10×0.5 ml fractions are appropriate however collecting morefractions which are thus smaller in volume is also possible (e.g.20×0.25 ml fractions). The fractions are stored at −80° C. untilanalysis. collected fractions are analysed to determine mitochondrialintegrity using any of the methods described herein (e.g., in Example10, 11) or known in the art.

Example 13 Agents that Increase NAD levels Increase Oocyte Production

As shown in FIG. 37, NAD⁺ is synthesized via three main pathways: (i)the NAD Salvage pathway (via NAMPT and NMNAT1-3 from nicotinamide, NAMto NMN to NAD⁺); (ii) from tryptophan, via the de novo pathway, viaquinolinic acid (FIG. 38); and (iii) from nicotinamide riboside, amolecule found in milk and other food products (FIG. 38). Untilrecently, NAD⁺ was regarded simply as a coenzyme, carrying electronsfrom one reaction to another. It has now been discovered that NAD⁺ is aprimary signal for low caloric intake, coordinating the activities ofmajor metabolic pathways, in large part, by stimulating the activity ofsirtuins. Two critical downstream mediators are SIRT1 (a nuclearsirtuin) and SIRT3 (a mitochondrial sirtuin), which act synergisticallyto increase respiration and fatty acid oxidation in heart and skeletalmuscle in response to fasting and exercise. In oogonical stem cells,SIRT1 controls the expression of a critical transcription factor thatregulates the differentiation of oogonial stem cells into oocytes.

During aging, however, NAD⁺ levels in the nucleus and mitochondriadecline, reducing the activity of these two sirtuins and severelycompromising mitochondrial function. NAD⁺ levels can be increased byincubating cells with a NAD precursor such as NMN (e.g. FIG. 39), byinjecting or otherwise delivering an NAD precursor to cells in vivo(FIG. 42), by increasing the expression of genes that synthesize NAD⁺e.g. NAMPT, NMNAT1-3; or by inhibiting NAD degradation, via PARPs orCD38 inhibition. (see FIG. 37CD38 inhibitors include, but are notlimited to those listed herein above in Tables 2A and 2B and aredescribed by Dong M. et al. Org. Biomol. Chem. 2011 (9): 3246-3257 andKellenberger E. et al. Bioorg Med Chem Lett. 2011 21(13): 3939-42, thecontents of which are incorporated herein by reference. Increasing NAD+in cells can be achieved by other methods, such as applying substratesfor the TCA cycle (e.g. Pyruvate, fatty acids) The following experimentswere carried out to determine whether genes and small molecules thatraise NAD⁺ levels and/or activate the sirtuins delay or reverse theeffects of aging and cell stress/damage on female fertility in vivo andin vitro.

Oogonial stem cells were isolated from dissociated ovaries using a FACSbased sorting protocol to purify OSCs free of contaminating oocytes (fordetails, see Example 1). Cells were maintained in culture mediumconsisted of minimum essential medium α (MEMα), 10% FBS, 1 mM sodiumpyruvate, 1 mM non-essential amino acids, 2 mM L-1-glutamine, 0.1 mMβ-mercaptoethanol (Sigma), 10 ng/ml-1 LIF (Millipore), 1× N-2 MAX MediaSupplement (R&D) 10 ng/ml EGF (Epidermal growth factor, Recombinanthuman; Gibco Division of Thermofisher Scientific, Waltham, Ma., USA), 40ng/ml human GDNF (glial cell line-derived neurotrophic factor; R&Dsystems), 1 ng/ml human bFGF (basic fibroblast growth factor; GibcoDivision of Thermofisher Scientific, Waltham, Mass., USA)

For all experiments 25,000 cells were plated in each well of a 24 wellplate. Cells were allowed to attach for twenty-four hours and then weretreated with NMN (β-Nicotinamide mononucleotide; Sigma). Unlessotherwise stated, NMN was added twice to the cells, first at twelvehours and then again at six hours prior to analysis (12+6 h).Mitochondrial DNA Copy number was analysed as follows. Total cellularDNA was isolated from cells at the indicated time points using DNeasyBlood & Tissue Kit (Qiagen) according to the manufacturer'sinstructions. Mitochondrial DNA copy number was quantified usingLIGHTCYCLER 480 SYBR® Green I Master (Roche Applied Science, Penzberg,Germany) using the following primers on a LIGHTCYCLER® 480 PCR machine(Roche Applied Science (Penzberg, Germany).

MT- ND2: F: AAGGGATCCCACTGCACATA (SEQ ID NO: 70) R: AGTCCTCCTCATGCCCCTAT(SEQ ID NO: 71) RPS18 Nuclear F: CCAGAGGTTGCATTTTCCCAAG (SEQ ID NO: 72)R: TAAGGCCGATAAGGCAAACGAA (SEQ ID NO: 73)Following treatment with NMN, NAD⁺/NADH levels were measured accordingto the manufacturer's instructions using the NAD/NADH Quantitation Kit(Biovision) Raising NAD⁺ levels in cells and in vivo dramaticallyincreased mitochondrial function and mitochondrial content, which isgenerally recognized as a major determinant of female fertility,metabolic health, brain function, cardiovascular health and glucosemetabolism/type II diabetes. OSCs and oocytes treated with an NADprecursor (e.g. nicotinamide riboside ie. “NMN”, see FIG. 38) hadincreased NAD⁺, NAD⁺:NADH, and mitochondrial DNA content (FIG. 40).

To determine whether increasing NAD⁺ levels had an effect on oocyteproduction, spontaneous oocyte formation was assayed. Each well of a 24-well plate was seeded with 25,000 OSCs. The number of oocytes formed andreleased into the medium per well was assessed the second day afterseeding as well as the designated time points after NMN treatment. NMNtreatment increased the rate of egg formation (EFA) (FIGS. 37, 38, and41). Based on these results, compounds and genes that increase NAD⁺ invivo or in vitro are expected to reduce or reverse infertilityassociated with mitochondrial damage, energetic defects, and aging ofthe ovary in female subjects. NMN treatment is also expected to enhancethe function of OCSsOSCs, oocytes, granulose cells, and blood vessels inthe ovary.

NMN and other compounds that increase NAD⁺ levels are useful forincreasing fertility or otherwise reducing or reversing infertility in afemale subject. In one embodiment, such compounds are delivered to asubject orally, by intraperitoneal injection (IP), or subcutaneously toincrease the probability that the subject will conceive and deliverhealthy offspring. Systemic administration of the NAD⁺ precursor NMNraised NAD⁺ levels in vivo in young and old mice. Cardiac [NAD⁻]declines with age. This decline in NAD⁺ was reversed by NMN treatment(n=3; 200 mg.kg.d. I.P. for 1 week)(FIG. 42). NMN treatment also had arestorative effect on mitochondrial function (FIG. 43). In anotherembodiment, NMN and other compounds that increase NAD⁺ levels aredelivered to an OSC, oocyte, blasotcyst, sperm, or isolated mitochondriain vitro. For example, such compounds are delivered to a germ cell priorto, during or following IVF. In another embodiment, a compound of theinvention is used to enhance the yield or preservation of mitochondriafrom an OSC, oocyte, blasotcyst, sperm, or isolated mitochondria invitro.

Example 14 Oral Intake of Apigenenin, Luteolin, and SRT1720 ImproveQuality of Oocytes in Aged Females

Female mice (8 months old, strain C57BL/6) were maintained on a 12:12light:dark cycle and provided ad-libitum access to water and food.Conditions within rooms were maintained at 21°±1° C. with 50%±20%relative humidity. Mice were placed on the experimental diets at 8.5months of age and were maintained for 3 months on the diets. All dietswere custom made, ordered from Research Diets: OpenStandard Diet (20kcal % Protein, 15 kcal % Fat and 65 kcal % Carbohydrate) and theexperimental groups consisted of regular OpenStandard Diet, OpenStandardDiet+Apigenin at 0.5 g/kg of body weight, OpenStandard Diet+Luteolin at0.5 g/kg, and OpenStandard Diet+SRT-1720 at 2 g/kg. Food intake wasmeasured weekly and average weight gain was assessed every two weeks.Each experimental group consisted of 12 randomly allocated mice. Whenmice were euthanized an additional group of 3-month old C57BL/6 females(3 M) were used as positive controls. In all groups, oocyte numbers wereassessed following hormonal stimulation for superovulation.

It was determined that 11.5 month control mice in this study ovulatevery few if any oocytes, and apigenenin, luteolin, and SRT-1720 allincrease oocyte yield (FIG. 44). The 12M control group fed OpenStandardDiet failed to ovulate a sufficient number of oocytes for subsequentanalyses (approximately 1.3 oocytes/mouse, n=12 mice). Because of theextremely low yield of oocytes from the aged control females, historicaldata for oocyte yield from 12-month-old C57BL/6 females (referred to as“Hist 12 M”; from Selesniemi et al., Proc Natl Acad Sci USA. 2011 July26; 108(30): 12319-12324) can be used as an additional reference pointfor an aged control group.

It was also determined that apigenenin, luteolin, and SRT-1720 do notaffect the percentage of mature, metaphase II oocytes retrievedfollowing superovulation as compared to age appropriate controls (Hist12 M). In FIG. 45A, the percentage of oocytes assessed at metaphase IIis shown. In FIG. 45B, the percentage of oocytes arrested at thegerminal vesicle (immature) stage is shown. In FIG. 45C, the percentageof atretic (dead) oocytes is shown.

It was also determined that apigenenin, luteolin, and SRT-1720 improvethe quality of oocytes in aged mice as compared to age appropriatecontrols (Hist 12 M). In FIG. 46A, it is shown that the percentage ofoocytes exhibiting abnormal mitochondrial clustering is reduced in micefed apigenin, luteolin, or SRT-1720 (methods are described in Selesniemiet al., Proc Natl Acad Sci USA. 2011 July 26; 108(30): 12319-12324). InFIGS. 46B and 46C respectively, it is shown that mice fed apigenenin,luteolin, or SRT-1720 have a reduced percentage of spindle abnormalitiesas well as reduced chromosomal misalignment in MII oocytes. Methods fordetermining spindle abnormalities and chromosomal misalignment aredescribed above in Example 8. Abnormal mitochondrial distribution wasdetermined to be present in 16.13% of oocytes from 3 M mice, 46% ofoocytes from Hist 12 M mice, 7.7% of oocytes following supplementationwith apigenenin, 12% of oocytes following supplementation with luteolinand 6.9% of oocytes following supplementation with SRT-1720. Spindleabnormalities were determined to be present in 18.3% of oocytes in 3 Mmice, 60% of oocytes in Hist 12 M mice, 32% of oocytes followingsupplementation with apigenenin, 32% of oocytes following upplementationwith luteolin and 32.8% of oocytes following supplementation withSRT-1720. Chromosomal misalignment was determined to be present 18.3% ofoocytes in 3 M mice, 62% of oocytes in Hist 12 M mice, 44% of oocytesfrom mice supplemented with apigenenin, 40% of oocytes followingsupplementation with luteolin and 40.9% of oocytes followingsupplementation with SRT-1720.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

What is claimed is:
 1. A composition comprising a mammalian oocyte, amammalian oogonial stem cell (OSC), the progeny of a mammalian OSC or amammalian preimplantation zygote, a CD38 inhibitor and a nicotinamideadenine dinucleotide (NAD precursor), wherein: the OSC is obtained fromovarian tissue, is an isolated non-embryonic stem cell, and ismitotically competent and expresses Vasa, Oct-4, Dazl and Stella and,optionally, a stage-specific embryonic antigen; the CD38 inhibitor is acompound selected from the group consisting of apigenin, luteolin,tyrphostin-8, berberine and SRT-1720; and the NAD precursor is acompound selected from the group consisting of nicotinamidemononucleotide, nicotinamide riboside or nicotinic acid; and wherein theCD38 inhibitor and the NAD precursor are present in the composition inan amount effective to enhance the bioenergetic status of said oocyte,OSC, progeny of an OSC or preimplantation zygote in said composition. 2.The composition of claim 1, further comprising a culture medium selectedfrom the group consisting of cell culture medium, oocyte retrievalsolution, oocyte washing solution, oocyte in vitro maturation medium,ovarian follicle in vitro maturation medium, oocyte in vitrofertilization medium, vitrification solution and cryopreservationsolution.
 3. The composition of claim 1, wherein the NAD precursor isnicotinamide riboside.
 4. The composition of claim 1, wherein the NADprecursor is nicotinic acid.
 5. The composition of claim 1, wherein amammalian OSC is cultured.
 6. The composition of claim 1, wherein themammalian oocyte, or OSC, progeny of an OSC or preimplantation zygote isfrom a human female.
 7. The composition of claim 6, wherein the humanfemale is selected from the group consisting of females of advancedmaternal age, females suffering from oocyte-related infertility andfemales with low ovarian reserve.
 8. The composition of claim 2, whereinthe CD38 inhibitor and the NAD precursor are present in the medium in anamount effective to increase the number of functional mitochondria inthe mammalian oocyte, OSC, progeny of an OSC or preimplantation zygotecultured in said medium.
 9. The composition of claim 2, wherein the CD38inhibitor and the NAD precursor are present in the medium in an amounteffective to increase the mitochondrial energy of the mammalian oocyte,OSC, progeny of an OSC or preimplantation zygote cultured in saidmedium.
 10. The composition of claim 2, wherein the CD38 inhibitor andthe NAD precursor are present in the medium in an amount effective toincrease the cellular energy of the mammalian oocyte, OSC, progeny of anOSC or preimplantation zygote cultured in said medium.
 11. Thecomposition of claim 1, wherein the CD38 inhibitor is present at aconcentration≥25 μM.
 12. The composition of claim 1, wherein the NADprecursor is present at a concentration≥100 μM.
 13. The composition ofclaim 2, wherein the medium further comprises ovarian tissue, ovarianfollicles, bone marrow, umbilical cord blood or peripheral blood. 14.The composition of claim 1, wherein the mammalian OSC expresses astage-specific embryonic antigen.